Crosslinked peptide hydrogels

ABSTRACT

The present invention relates to hydrogels comprising a plurality of amphiphilic peptides and/or peptoids capable of self-assembling into three-dimensional macromolecular nanofibrous networks, which entrap water and form said hydrogels, wherein at least a portion of said plurality of amphiphilic peptides and/or peptoids is chemically cross-linked. The present invention further relates to methods for preparing such hydrogels and to various uses of such hydrogels, e.g. as cell culture substrates, for drug and gene delivery, as wound dressing, as an implant, as an injectable agent that gels in situ, in pharmaceutical or cosmetic compositions, in regenerative medicine, in tissue engineering and tissue regeneration, or in electronic devices. It also relates to a method of tissue regeneration or tissue replacement using a hydrogel in accordance with the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/634,559, entitled “CROSSLINKED PEPTIDE HYDROGELS” and filedon Feb. 27, 2015, which is a divisional application of U.S. patentapplication Ser. No. 13/751,295, entitled “CROSSLINKED PEPTIDEHYDROGELS”, and filed on Jan. 28, 2013, now U.S. Pat. No. 8,999,916,which is a continuation in part application of U.S. patent applicationSer. No. 13/638,152, entitled “AMPHIPHILIC LINEAR PEPTIDE/PEPTOID ANDHYDROGEL COMPRISING THE SAME”, and filed on Sep. 28, 2012, now U.S. Pat.No. 9,067,084, which is a 35 U.S.C. § 371 National Stage ofInternational Application No. PCT/SG2010/000469, entitled “AMPHIPHILICLINEAR PEPTIDE/PEPTOID AND HYDROGEL COMPRISING THE SAME” and filed onDec. 15, 2010 and claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/319,838, filed Mar. 31, 2010, the entirecontents of each of which are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to hydrogels comprising a plurality ofamphiphilic peptides and/or peptoids capable of self-assembling intothree-dimensional macromolecular nanofibrous networks, which entrapwater and form said hydrogels, wherein at least a portion of saidplurality of amphiphilic peptides and/or peptoids is chemicallycross-linked. The present invention further relates to methods forpreparing such hydro gels and to various uses of such hydrogels, e.g. ascell culture substrates, for drug and gene delivery, as wound dressing,as an implant, as an injectable agent that gels in situ, inpharmaceutical or cosmetic compositions, in regenerative medicine, intissue engineering and tissue regeneration, or in electronic devices. Italso relates to a method of tissue regeneration or tissue replacementusing a hydrogel in accordance with the present invention.

SEQUENCE LISTING

In accordance with 37 CFR 1.52(e)(5), the present specification makesreference to a Sequence Listing submitted electronically in the form ofan ASCII text file (entitled “Sequence_Listing.txt”, created on May 18,2015 and 11 KB in size). The entire contents of the Sequence Listing areherein incorporated by reference, with the intention that, uponpublication (including issuance), this incorporated sequence listingwill be inserted into the published document immediately before theclaims.

BACKGROUND OF THE INVENTION

Supramolecular structures are held together by intermolecular bondingsthat are responsible for the organization of polymolecular systems. Thenon-covalent, intermolecular forces which are required for the assemblyof the defined supramolecular structures are mainly electrostaticinteractions, hydrogen bondings, van der Waals forces, etc.Supramolecular chemistry or biology gathers a vast body of two or threedimensional complex structures and entities formed by association ofchemical or biological species. These associations are governed by theprinciples of molecular complementarity or molecular recognition andself-assembly. The knowledge of the rules of intermolecular associationcan be used to design polymolecular assemblies in form of membranes,films, layers, micelles, tubules, gels for a variety of biomedical ortechnological applications (J.-M. Lehn, Science, 295, 2400-2403, 2002).

Peptides have been used for the fabrication of supramolecular structuresthrough molecular self-assembly (S. Zhang, Nature Biotechnology, 21,1171-1178, 2003). Peptides are for instance able to assemble intonanotubes (US 7, 79, 84) or into supramolecular hydrogels consisting ofthree dimensional scaffolds with a large amount of around 98-99%immobilized water or aqueous solution. The peptide-based biomaterialsare powerful tools for potential applications in biotechnology, medicineand even technical applications. Depending on the individual propertiesthese peptide-based hydrogels are thought to serve in the development ofnew materials for tissue engineering, regenerative medicine, as drug andvaccine delivery vehicles or as peptide chips for pharmaceuticalresearch and diagnosis (E. Place et al., Nature Materials, 8, 457-470,2009). There is also a strong interest to use peptide-basedself-assembled biomaterial such as gels for the development of molecularelectronic devices (A. R. Hirst et al., Angew. Chem. Int. Ed., 47,8002-8018, 2008).

A variety of “smart peptide hydro gels” have been generated that reacton external manipulations such as temperature, pH, mechanical influencesor other stimuli with a dynamic behavior of swelling, shrinking ordecomposing. Nevertheless, these biomaterials are still not “advanced”enough to mimic the biological variability of natural tissues as forexample the extracellular matrix (ECM) or cartilage tissue or others.The challenge for a meaningful use of peptide hydrogels is to mimic thereplacing natural tissues not only as “space filler” or mechanicalscaffold, but to understand and cope with the biochemical signals andphysiological requirements that keep the containing cells in the rightplace and under “in vivo” conditions (R. Fairman and K. Akerfeldt,Current Opinion in Structural Biology, 15, 453-463, 2005). Much efforthas been undertaken to understand and control the relationship betweenpeptide sequence and structure for a rational design of suitablehydrogels. In general hydrogels contain macroscopic structures such asfibers that entangle and form meshes. Most of the peptide basedhydrogels utilize as their building blocks 0-pleated sheets whichassemble to fibers. Later it was shown that it is possible to designhydrogelating self-assembling fibers purely from a-helices. Besides0-sheet structure-based materials (S. Zhang et al., PNAS, 90, 3334-3338,1993; A. Aggeli et al., Nature, 386, 259-262, 1997, etc.) a variety ofa-helical hydrogels has been developed (W. A. Petka et al., Science,281, 389-392, 1998; C. Wang et al., Nature, 397, 417-420, 1999; C.Gribbon et al., Biochemistry, 47, 10365-10371, 2008; E. Banwell et al.,Nature Materials, 8, 596-600, 2009, etc.).

Nevertheless, the currently known peptide hydrogels are in most of thecases associated with low rigidity, sometimes unfavourable physiologicalproperties and/or complexity and the requirement of substantialprocessing thereof which leads to high production costs. There istherefore a widely recognized need for peptide hydrogels that are easilyformed, non-toxic and have a sufficiently high rigidity for standardapplications. The hydrogels should also be suitable for the delivery ofbioactive moieties (such as nucleic acids, small molecule therapeutics,cosmetic and anti-microbial agents) and/or for use as biomimeticscaffolds that support the in vivo and in vitro growth of cells andfacilitate the regeneration of native tissue.

SUMMARY OF THE INVENTION

It is therefore desirable to provide a biocompatible compound that iscapable of forming a hydrogel that meets at least some of the aboverequirements to a higher extent than currently available hydro gels andthat is not restricted by the above mentioned limitations.

Disclosed is an amphiphilic peptide and/or peptoid capable of forming ahydrogel, the amphiphilic peptide and/or peptoid comprising anamphiphilic sequence consisting of:

a hydrophobic sequence stretch of n aliphatic amino acids, wherein n isan integer from 2 to 15, and

a hydrophilic sequence stretch linked to said hydrophobic sequencestretch and having a polar moiety which is acidic, neutral or basic,said polar moiety comprising m adjacent hydrophilic amino acids, whereinm is an integer from 1 to 5.

In one embodiment the amphiphilic peptide and/or peptoid has aC-terminus and an N-terminus wherein the N-terminus is protected by aprotecting group, wherein said protecting group preferably is an acetylgroup.

In one embodiment, the amphiphilic peptide and/or peptoid has aC-terminus, which, if a basic polar amino acid is located at theC-terminus, is preferably amidated.

In one embodiment, n is an integer from 2 to 6.

In one embodiment, m is an integer from 1 to 2.

In one embodiment, the amphiphilic peptide and/or peptoid consists of oamphiphilic sequences, as defined above, which amphiphilic sequences arelinked to each other, o being an integer from 1 to 50.

In one embodiment, for a given amphiphilic peptide and/or peptoid, saidaliphatic amino acids and said hydrophilic amino acids are eitherD-amino acids or L-amino acids.

In one embodiment, each of the hydrophilic amino acids has a polar groupwhich is independently selected from a hydroxyl, an ether, a carboxyl,an imido, an amido, an ester, an amino, a guanidine, a thio, athioether, a seleno, and a telluro group.

In one embodiment, said polar moiety of said hydrophilic sequencestretch comprises m adjacent hydrophilic amino acids, m being defined asdefined above, said hydrophilic amino acids being selected from thegroup comprising aspartic acid, asparagine, glutamic acid, glutamine,5-N-ethyl-glutamine (theanine), citrulline, thio-citrulline, cysteine,homocysteine, methionine, ethionine, selenomethionine,telluromethionine, threonine, allo-threonine, serine, homoserine,arginine, homoarginine, ornithine, lysine and N(6)-carboxymethyllysine,histidine, and wherein said hydrophobic sequence stretch comprises naliphatic amino acids, n being as defined above, said aliphatic aminoacids being selected from the group comprising isoleucine, norleucine,leucine, valine, alanine, glycine, homoallylglycine andhomopropargylglycine. In one embodiment, m is 1 to 2.

In one embodiment, m is 2 and said polar moiety comprises two identicalamino acids, or m is 1 and said polar moiety comprises any one ofaspartic acid, asparagine, glutamic acid, glutamine, serine, threonine,cysteine, methionine, lysine and histidine.

In one embodiment, said polar moiety is adjacent to the hydrophobicsequence stretch of n aliphatic amino acids.

In one embodiment, said polar moiety has a sequence selected from Asp,Asn, Glu, Gin, Ser, Thr, Cys, Met, Lys, His, Asn-Asn, Asp-Asp, Glu-Glu,Gln-Gln, Asn-Gln, Gln-Asn, Asp-Gln, Gin-Asp, Asn-Glu, Glu-Asn, Asp-Glu,Glu-Asp, Gln-Glu, Glu-Gln, Asp-Asn, Asn-Asp Thr-Thr, Ser-Ser, Thr-Ser,Ser-Thr, Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu Ser,Ser-Glu, Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr, Thr-Gln, Glu-Thr,Thr-Glu.

In one embodiment, said polar moiety comprises the C-terminus of theamphiphilic peptide and/or peptoid, or wherein said polar moietycomprises the N-terminus of the amphiphilic peptide and/or peptoid.

In one embodiment, both said C-terminus and said N-terminus do not carryany protecting groups attached to them.

In one embodiment, said polar moiety comprises the C-terminus of theamphiphilic peptide and/or peptoid, wherein both said C-terminus andsaid N-terminus do not carry any protecting groups attached to them.

In one embodiment, said polar moiety comprises the C-terminus of theamphiphilic peptide and/or peptoid, wherein said C-terminus does notcarry any protecting group, and wherein said N-terminus carries aprotecting group.

In one embodiment, said protecting group is an acetyl group attached tothe amino-group of said N-terminus.

In one embodiment, said polar moiety comprises the C-terminus of theamphiphilic peptide and/or peptoid, wherein said C-terminus carries aprotecting group, and wherein said N-terminus does not carry anyprotecting group.

In one embodiment, said protecting group is an amido-group attached tothe carboxyl group of said C-terminus.

In one embodiment, said polar moiety comprises the C-terminus of theamphiphilic peptide and/or peptoid, wherein both said C-terminus andN-terminus carry a protecting group.

In one embodiment, said C-terminus protecting group is an amido-groupattached to the carboxyl group of said C-terminus, and wherein saidN-terminus protecting group is an acetyl group attached to theamino-group of said N-terminus.

In one embodiment, said polar moiety consists of at least one amino acidpositioned at the C-terminus of the amphiphilic peptide and/or peptoid.

In one embodiment, said hydrophobic sequence stretch comprises and/orforms the N-terminus of the amphiphilic peptide and/or peptoid.

In one embodiment, all or a portion of the aliphatic amino acids of thehydrophobic sequence stretch are arranged in an order of decreasingamino acid size in the direction from N- to C-terminus of theamphiphilic peptide and/or peptoid, wherein the size of the aliphaticamino acids is defined as I=L>V>A>G.

In one embodiment, said aliphatic amino acids arranged in an order ofdecreasing amino acid size have a sequence which is a repetitive ornon-repetitive sequence.

In one embodiment, said aliphatic amino acids arranged in order ofdecreasing amino acid size have a sequence with a length of 2 to 7,preferably 2 to 6, more preferably 2 to 5 amino acids. In oneembodiment, said aliphatic amino acids arranged in an order ofdecreasing amino acid size have a sequence selected from LIVAG, ILVAG,LIVAA, LAVAG, IVAG, LIVA, LIVG, IVA and IV, wherein, optionally, thereis an A preceding such sequence at the N-terminus.

In one embodiment, all or a portion of the aliphatic amino acids of thehydrophobic sequence stretch are arranged in an order of identical aminoacid size in the amphiphilic peptide and/or peptoid.

In one embodiment, said aliphatic amino acids arranged in order ofidentical amino acid size have a sequence with a length of 2 to 4 aminoacids. In one embodiment, said aliphatic amino acids arranged in anorder of identical size have a sequence selected from LLLL, LLL, LL,IIII, III, II, VVVV, VVV, VV, AAAA, AAA, AA, GGGG, GGG, and GG.

In one embodiment, the amphiphilic sequence undergoes a conformationalchange during self-assembly, preferably a conformational change from arandom coil conformation to a helical intermediate structure to a finalbeta conformation. In one embodiment, the conformational change isconcentration dependent.

In one embodiment, the amphiphilic linear sequence comprises a singlehydrophilic and at least two aliphatic amino acids.

In one embodiment, the amphiphilic sequence is one of SEQ ID NO: 1-42.It should be noted that any of the amphiphilic sequences may carry aprotecting group at the N-terminus or the C-terminus or both. Forexample, SEQ ID NO:1-42 may all carry an acetyl group as protectinggroup at the N-terminus. As a further example, SEQ ID NO: 19 (LIVAGK)may carry an amido-group as protecting group at the C-terminus, andadditionally it may have an acetyl group at the N-terminus as protectinggroup.

In one embodiment, said amphiphilic peptide and/or peptoid is stable inaqueous solution at physiological conditions at ambient temperature fora period of time in the range from 1 day to at least 6 months,preferably to at least 8 months more preferably to at least 12 months.

In one embodiment, the amphiphilic peptide and/or peptoid is stable inaqueous solution at physiological conditions, at a temperature up to 90°C., for at least 1 hour.

Also disclosed is a hydrogel comprising the amphiphilic peptide and/orpeptoid as defined above.

In one embodiment, the hydrogel is stable in aqueous solution at ambienttemperature for a period of at least 7 days, preferably at least 2 to 4weeks, more preferably at least 1 to 6 months.

In one embodiment, the hydrogel is characterized by a storage modulus G′to loss modulus G″ ratio that is greater than 2.

In one embodiment, the hydrogel is characterized by a storage modulus G′from 100 Pa to 80, 00 Pa at a frequency in the range of from 0.02 Hz to16 Hz.

In one embodiment, the hydrogel has a higher mechanical strength thancollagen or its hydrolyzed form (gelatin).

In one embodiment, the hydrogel as defined above comprises fibers of theamphiphilic peptide and/or peptoid as defined above, said fibersdefining a network that is capable of entrap-ping at least one of amicroorganism, a virus particle, a peptide, a peptoid, a protein, anucleic acid, an oligosaccharide, a polysaccharide, a vitamin, aninorganic molecule, a synthetic polymer, a small organic molecule or apharmaceutically active compound.

In one embodiment, the hydrogel comprises at least one of amicroorganism, a virus particle, a peptide, a peptoid, a protein, anucleic acid, an oligosaccharide, a polysaccharide, a vitamin, aninorganic molecule, a synthetic polymer, a small organic molecule or apharmaceutically active compound entrapped by the network of fibers ofthe amphiphilic polymer.

In one embodiment, the fibers of the amphiphilic polymer are coupled tothe at least one of a microorganism, a virus particle, a peptide, apeptoid, a protein, a nucleic acid, an oligosaccharide, apolysaccharide, a vitamin, an inorganic molecule, a synthetic polymer, asmall organic molecule or a pharmaceutically active compound entrappedby the network of fibers of the amphiphilic polymer.

In one embodiment, the hydrogel is comprised in at least one of a fuelcell, a solar cell, an electronic cell, a biosensing device, a medicaldevice, an implant, a pharmaceutical composition and a cosmeticcomposition. In one embodiment, the hydrogel as defined above is for usein at least one of the following:

release of a pharmaceutically active compound, medical tool kit, a fuelcell, a solar cell, an electronic cell, tissue regeneration, stem celltherapy and gene therapy.

In one embodiment, the hydrogel as defined above is injectable.

Also disclosed is a method of preparing a hydrogel, the methodcomprising dissolving an amphiphilic peptide and/or peptoid as definedabove in an aqueous solution.

In one embodiment, the dissolved amphiphilic peptide and/or peptoid inaqueous solution is further exposed to temperature, wherein thetemperature is in the range from 20° C. to 90° C., preferably from 20°C. to 70° C.

In one embodiment, the amphiphilic peptide and/or peptoid is dissolvedat a concentration from 0.01 μg/ml to 100 mg/ml, preferably at aconcentration from 1 mg/ml to 50 mg/ml, more preferably at aconcentration from about 1 mg/ml to about 20 mg/ml.

Also disclosed is a surgical implant, or stent, the surgical implant orstent comprising a peptide and/or peptoid scaffold, wherein the peptideand/or peptoid scaffold is formed by a hydrogel as defined above.

Also disclosed is a pharmaceutical and/or cosmetic composition and/or abiomedical device and/or electronic device comprising the amphiphilicpeptide and/or peptoid as defined above.

In one embodiment, the pharmaceutical and/or cosmetic composition and/orthe biomedical device, and/or the electronic devices as defined above,further comprises a pharmaceutically active compound.

In one embodiment, the pharmaceutical and/or cosmetic composition asdefined above, further comprises a pharmaceutically acceptable carrier.

Also disclosed is a kit of parts, the kit comprising a first containerwith an amphiphilic peptide and/or peptoid as defined above and a secondcontainer with an aqueous solution.

In one embodiment, the aqueous solution of the second container furthercomprises a pharmaceutically active compound.

In one embodiment, the first container with an amphiphilic peptideand/or peptoid further comprises a pharmaceutically active compound.

Also disclosed is a method of tissue regeneration comprising the steps:

providing a hydrogel as defined above, exposing said hydrogel to cellswhich are to form regenerated tissue, allowing said cells to grow onsaid hydrogel.

In one embodiment, the method as defined above is performed in-vitro orin-vivo.

In one embodiment, the method as defined above is performed in vivo,wherein, in step a), aid hydrogel is provided at a place in a body wheretissue regeneration is intended.

In one embodiment, said step a) is performed by injecting said hydrogelat a place in the body where tissue regeneration is intended.

In a first aspect the present disclosure provides an amphiphilic peptideand/or peptoid capable of forming a hydrogel. The amphiphilic peptideand/or peptoid includes a hydrophobic and a hydrophilic sequence. Thishydrophobic sequence has a length of n L- or D-amino acids. n is aninteger, which may typically range from 2 to about 15. The hydrophilicsequence has a polar and/or charged moiety comprising m L- or D-aminoacids. m is an integer from 1 to 5. Each of the m aliphatic amino acidscarries an independently selected polar group. The amphiphilic linearsequence has a net charge at physiological pH and a N-terminus carryinga protecting group. The protecting group can be an acetyl group. Theamphiphilic peptide and/or peptoid may comprise o linked amphiphilicpeptide and/or peptoid sequences of n hydrophobic and m hydrophilic L-and D-amino acids, wherein o is an integer from 1 to about 50. Theamphiphilic peptide and/or peptoid may consist of o linked amphiphilicpeptide and/or peptoid sequences of n hydrophobic and m hydrophilic L-and D-amino acids. The value of n may be an integer from 2 to about 15.The value of m may be 1 to 5. The charged and/or polar group of each ofthe m hydrophilic L- and D-amino acids may be independently selectedfrom a hydroxyl, an ether, a carboxyl, an amido, an ester, an amino, aguanidino, athio, a thioether, a seleno, and a telluro group. Thecharged or polar moiety of the hydrophilic sequence may comprise m L- orD-amino acids selected from the group consisting of aspartic acid,asparagine, glutamic acid, glutamine, 5-N-ethyl-glutamine (theanine),citrulline, thiocitrulline, cysteine, homocysteine, methionine,ethionine, selenomethionine, telluromethionine, threonine,allo-threonine, serine, homoserine, arginine, homoarginine, ornithine,lysin and N(6)-carboxymethyllysine. The charged and/or polar moiety ofthe hydrophilic sequence may comprise two identical amino acids. The twoidentical amino acids may be adjacent to the non-polar hydrophobicmoiety. The charged and/or polar moiety may consist of two amino acidswith a sequence selected from Asn-Asn, Asp-Asp, Glu-Glu, Gln-Gln,Asn-Gln, Gln-Asn, Asp-Gin, Gin-Asp, Asn-Glu, Glu-Asn, Asp-Glu, Glu-Asp,Gln-Glu, Glu-Gln, Asp-Asn, Asn-Asp, Thr-Thr, Ser-Ser, Thr-Ser, Ser-Thr,Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser, Ser-Gln, Glu-Ser, Ser-Glu,Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr, Thr-Gln, Glu-Thr, Thr-Glu.The charged and/or polar moiety may comprise the C-terminus of theamphiphilic peptide and/or peptoid. The charged and/or polar moiety maycomprise (i) the C-terminus, the C-terminus carrying an unprotectedC-terminal carboxyl group or (ii) the N-terminus, the N-terminuscarrying an unprotected N-terminal amino group. The charged and/or polarmoiety may comprise the C-terminus of the amphiphilic peptide and/orpeptoid, the C-terminus carrying an unprotected C-terminal carboxylgroup and wherein the N-terminus carries a protecting group preferablythe acetyl group. The protecting group may be an amido protecting group.The charged and/or polar moiety may consist of at least one amino acidpositioned at the C-terminus of the amphiphilic peptide and/or peptoid.The hydrophobic sequence may comprise at least two aliphatic amino acidsthat is defined by a main chain comprising 1 to about 20 carbon atoms. Aportion of the amino acids of the non-polar moiety may be arranged in ageneral sequence of decreasing size in the direction from N- toC-terminus of the amphiphilic peptide and/or peptoid, and the size ofadjacent amino acids of the non-polar moiety may be identical or smallerin the direction of the general sequence of decreasing size. The generalsequence of decreasing size may be preferably a non-repetitive sequence.The direction of the general sequence of decreasing size in whichadjacent amino acids may be of identical or smaller size may be thedirection toward the charged and/or polar moiety of the sequence. Theportion of the amino acids arranged in a general sequence of decreasingsize may have a length of 2-7, preferably 2-6, more preferably 2, 3, 4,5 or 6 amino acids. The portion of the amino acids arranged in a generalsequence of decreasing size may also have a length of n−m−1 acids andwherein the portion of the amino acids arranged in the general sequenceof decreasing size may be positioned between the remaining non-polaramino acid of the non-polar moiety of n−m amino acids and the polarmoiety. The remaining nonpolar amino acid of the non-polar moiety of n−mamino acids may define the N-terminus or the C-terminus of theamphiphilic peptide and/or peptoid. The remaining non-polar amino acidof the non-polar moiety of n−m amino acids may be one of alanine, valineand glycine. The amphiphilic linear sequence may undergo aconformational change from a random coil conformation to a helicalconformation during self-assembly. The conformational change may beconcentration dependent. The non-polar moiety of the amphiphilic linearsequence may comprise at least one L- or D-amino acid selected from thegroup consisting of glycine, homoallylglycine, homopropargylglycine,alanine, valine, leucine, norleucine and isoleucine. The amphiphiliclinear sequence may comprise a single polar and/or charge and a singlenonpolar moiety. The amphiphilic linear sequence may have a positive ora negative net charge. The net charge may be from about −1 to about −4or from about +5 to about +1. The net charge may be from about −1 toabout −2. The net charge may be −2. The net charge may be +1 or +2 or+5. The amphiphilic peptide and/or peptoid may be stable in aqueoussolution at physiological conditions at ambient temperature for a periodof time in the range from 1 day to at least 6 months, preferably atleast 8 months, more preferably at least 12 months. The amphiphilicpeptide and/or peptoid may be stable in aqueous solution atphysiological conditions at a temperature to 90° C. for at least 1 hour.

In a second aspect the disclosure provides a hydrogel. The hydrogelincludes an amphiphilic peptide and/or peptoid according to the firstaspect. The hydrogel may be stable in aqueous solution at ambienttemperature for a period of at least 7 days. The hydrogel may be stablein aqueous solution at ambient temperature for a period of at least 2 to4 weeks. The hydrogel may be stable in aqueous solution at ambienttemperature for a period of at least 1 to 6 months. The hydrogelmechanical property may be characterized by a loss modulus G″ to storagemodulus G′ ratio that is less than 1. The hydrogel may be characterizedby magnitude of storage modulus G′ greater than loss modulus G″ byminimum factor of 1.5. The hydrogel may be characterized by a storagemodulus G′ of from 100 Pa to 80, 00 Pa at a frequency in the range offrom 0.02 Hz to 16 Hz. The hydrogel may be characterized by higherstorage modulus G′ with increase in the concentration of peptide. Thehydrogel may have a higher mechanical strength than collagen orhydrolyzed form (gelatin). The hydrogel may comprise fibers of anamphiphilic peptide and/or peptoid described herein. The fibers maydefine a network that is capable of entrapping at least one of amicroorganism, a virus particle, a peptide, a peptoid, a protein, anucleic acid, an oligosaccharide, a polysaccharide, a vitamin, aninorganic molecule, a synthetic polymer, a small organic molecule or apharmaceutically active compound. The hydrogel may comprise at least oneof a microorganism, a virus particle, a peptide, a peptoid, a protein, anucleic acid, an oligosaccharide, a polysaccharide, a vitamin, aninorganic molecule, a synthetic polymer, a small organic molecule or apharmaceutically active compound entrapped by the network of fibers ofthe amphiphilic polymer. The fibers of the amphiphilic polymer may becoupled to the at least one of a microorganism, a virus particle, apeptide, a peptoid, a protein, a nucleic acid, an oligosaccharide, apolysaccharide, a vitamin, an inorganic molecule, a synthetic polymer, asmall organic molecule or a pharmaceutically active compound entrappedby the network of fibers of the amphiphilic polymer. The hydrogel may becomprised in at least one of a fuel cell, a solar cell, a electroniccell, a biosensing device, a medical device, an implant, apharmaceutical composition, drug delivery system, tissue culture medium,biosensor devices and a cosmetic composition. The hydrogel may be for atleast one of release of a pharmaceutically active compound, medical toolkit, a fuel cell, a solar cell, an electronic cell, tissue regeneration,stem cell therapy and gene therapy. In some embodiments the hydrogel maybe used for tissue regeneration, drug release or gene therapy.

In a third aspect the disclosure provides a method of preparing ahydrogel. The method includes providing an amphiphilic peptide and/orpeptoid according to the first aspect. The method further includesdissolving and/or dispersing the amphiphilic peptide and/or peptoid inan aqueous solution. The dissolved/dispersed amphiphilic peptide and/orpeptoid in aqueous solution may be further exposed to a temperature. Thetemperature may be selected in the range from about 20° C. to about 90,preferably from 20° C. to 70° C. The amphiphilic peptide and/or peptoidmay be dissolved at a concentration from about 0.01 μg/ml to about 100mg/ml. The amphiphilic peptide and/or peptoid may be dissolved at aconcentration from about 1 mg/ml to about 50 mg/ml. The amphiphilicpeptide and/or peptoid may be dissolved and/or dispersed at aconcentration from about 1 mg/ml to about 30 mg/ml.

In a fourth aspect the disclosure provides a surgical implant or stent.The surgical implant or stent includes a peptide and/or peptoidscaffold. The peptide and/or peptoid scaffold is defined by a hydrogelaccording to the second aspect.

In a fifth aspect the disclosure provides a pharmaceutical and/orcosmetic composition. The pharmaceutical and/or cosmetic compositionincludes the amphiphilic peptide and/or peptoid according to the firstaspect. The pharmaceutical and/or cosmetic composition may comprise apharmaceutically active compound. The pharmaceutical and/or cosmeticcomposition may comprise a pharmaceutically acceptable carrier.

In a sixth aspect the disclosure provides a kit of parts. The kitincludes a first container and a second container. The first containerincludes a peptide and/or peptoid according to the first aspect. Thesecond container includes an aqueous solution. The aqueous solution ofthe second container may further comprise a pharmaceutically activecompound. The first container with an amphiphilic peptide and/or peptoidmay further comprise a pharmaceutically active compound.

It was an object of the present invention to further improve the abovedisclosed hydrogels in terms of their material properties, such asstiffness, elasticity and resistance to degradation. It was a furtherobject of the present invention to facilitate the conjugation ofbioactive agents or other compounds of interest (e.g. nanoparticles) tothe hydrogel. Yet another object was to reduce the amount of amphiphilicpeptides and/or peptoids required for preparing hydrogels as disclosedabove.

The objects of the present invention are solved by a hydrogel comprisinga plurality of amphiphilic peptides and/or peptoids capable ofself-assembling into three-dimensional macromolecular nanofibrousnetworks, which entrap water and form said hydrogel, the amphiphilicpeptides and/or peptoids having the general formula:Z_(p)—(X)_(n)—(Y)_(m)-AA_(thiol)-Z′q,wherein

Z is an N-terminal protecting group, X is, at each occurrence,independently selected from an aliphatic amino acid, Y is, at eachoccurrence, independently selected from a hydrophilic amino acid,AA_(thiol) is an amino acid comprising a thiol group, Z′ is a C-terminalprotecting group, n is an integer selected from 2 to 6, preferably 2 to5, m is selected from 0, 1 and 2, preferably 0 and 1, and p and q areindependently selected from 0 and 1, wherein, preferably, p is 1,wherein at least a portion of said plurality of amphiphilic peptidesand/or peptoids is chemically (e.g. covalently) cross-linked.

In one embodiment, said amino acid comprising a thiol group is selectedfrom cysteine and homocysteine.

In one embodiment, said at least a portion of said plurality ofamphiphilic peptides and/or peptoids is chemically cross-linked viasulfhydryl-to-sulfhydryl cross-linking (i.e. via disulfide bridges), viasulfhydryl-to-hydroxyl cross-linking, via sulfhydryl-to-aldehydecross-linking, via sulfhydryl-to-amine cross-linking, via peptidoglycansor via photo-induced cross-linking, preferably viasulfhydryl-to-sulfhydryl cross-linking. In one embodiment, saidN-terminal protecting group has the general formula —C(O)—R, wherein Ris selected from the group consisting of H, unsubstituted or substitutedalkyls, and unsubstituted or substituted aryls. Preferred alkyls aremethyl, ethyl, butyl, isobutyl, propyl and isopropyl.

In one embodiment, said N-terminal protecting group is an acetyl group(R=methyl).

In one embodiment, said N-terminal protecting group is a peptidomimeticmolecule, including natural and synthetic amino acid derivatives,wherein the N-terminus of said peptidomimetic molecule may be modifiedwith a functional group selected from the group consisting of carboxylicacid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog,thiol, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone,azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, thioester;aryl, ketone, sulphite, nitrite, phosphonate and silane.

In one embodiment, said C-terminal protecting group is an amide group.

In one embodiment, the C-terminus of said amphiphilic peptides and/orpeptoids has the formula —CONHR or —CONRR′, with R and R′ being selectedfrom the group consisting of H, unsubstituted or substituted alkyls, andunsubstituted or substituted aryls. Preferred alkyls are methyl, ethyl,butyl, isobutyl, propyl and isopropyl.

In one embodiment, said C-terminal protecting group is an ester group.

In one embodiment, the C-terminus of said amphiphilic peptide and/orpeptoid has the formula —CO₂R, with R being selected from the groupconsisting of H, unsubstituted or substituted alkyls, and unsubstitutedor substituted aryls. Preferred alkyls are methyl, ethyl, butyl,isobutyl, propyl and isopropyl.

In one embodiment, said C-terminal protecting group is a peptidomimeticmolecule, including natural and synthetic amino acid derivatives,wherein the C-terminus of said peptidomimetic molecule may be modifiedwith a functional group selected from the group consisting of carboxylicacid, amide, alcohol, aldehyde, amine, imine, nitrile, an urea analog,thiol, phosphate, carbonate, sulfate, nitrate, maleimide, vinyl sulfone,azide, alkyne, alkene, carbohydrate, imide, peroxide, ester, thioester,aryl, ketone, sulphite, nitrite, phosphonate and silane.

In one embodiment, for a given amphiphilic peptide and/or peptoid, saidaliphatic amino acid, said hydrophilic amino acid and said amino acidcomprising a thiol group are either D-amino acids or L-amino acids.

In one embodiment, said hydrophilic amino acid has a polar group whichis independently selected from a hydroxyl, an ether, a carboxyl, animido, an amido, an ester, an amino, a guanidino, a thio, a thioether, aseleno, and a telluro group.

In one embodiment, said hydrophilic amino acid is selected from thegroup consisting of aspartic acid, asparagine, glutamic acid, glutamine,5-N-ethyl-glutamine (theanine), citrulline, thio-citrulline, cysteine,homocysteine, methionine, ethionine, selenomethionine,telluromethionine, threonine, allo-threonine, serine, homoserine,arginine, homoarginine, ornithine (Om), 2,-diaminobutyric acid (Dab),2,-diaminopropionic acid (Dap), lysine and N(6)carboxymethyllysine andhistidine.

In one embodiment, said hydrophilic amino acid is selected from thegroup consisting of aspartic acid, asparagine, glutamic acid, glutamine,serine, threonine, cysteine, methionine, lysine, ornithine (Om),2,-diaminobutyric acid (Dab), 2,-diaminopropionic acid (Dap) andhistidine.

In one embodiment, said aliphatic amino acid is selected from the groupconsisting of isoleucine, norleucine, leucine, valine, alanine, glycine,homoallylglycine and homopropargylglycine. Preferably, said aliphaticamino acid is selected from the group consisting of isoleucine, leucine,valine, alanine and glycine.

In one embodiment, all or a portion of the aliphatic amino acids of theamphiphilic peptides and/or peptoids, i.e. (X)n, are arranged in anorder of decreasing amino acid size in the direction from N- toC-terminus of the amphiphilic peptides and/or peptoids, wherein the sizeof the aliphatic amino acids is defined as I=L>V>A>G.

In one embodiment, said aliphatic amino acids arranged in an order ofdecreasing amino acid size have a sequence which is a repetitive ornon-repetitive sequence. In one embodiment, said aliphatic amino acidsarranged in an order of decreasing amino acid size have a sequenceselected from LIVAG, ILVAG, LIVAA, LAVAG, LAVAG, LIVA, LIVG, IVA and IV,wherein, optionally, there is an A preceding such sequence at theN-terminus.

In one embodiment, said amphiphilic peptides and/or peptoids undergo aconformational change during self-assembly, preferably a conformationalchange from a random coil conformation to a helical intermediatestructure to a final beta conformation.

In one embodiment, the conformational change is dependent on theconcentration of the amphiphilic peptides and/or peptoids, dependent onthe ionic environment, pH dependent and/or temperature dependent. In oneembodiment, said amphiphilic peptides and/or peptoids are the same ordifferent. In one embodiment, (X)_(n)-(Y)_(m) is selected from the groupconsisting of SEQ ID NO: 1 to 42. In one embodiment,(X)_(n)-(Y)_(m)-AA_(thiol) is selected from the group consisting ofLIVAGKC (SEQ ID NO:43), LIVAGSC (SEQ ID NO: 44), LIVAGDC (SEQ ID NO:45), ILVAGKC (SEQ ID NO: 46), ILVAGDC (SEQ ID NO: 47), LIVAGC (SEQ IDNO: 48), AIVAGC (SEQ ID NO: 49), ILVAGC (SEQ ID NO: 50), IVKC (SEQ IDNO: 51), IVDC (SEQ ID NO: 52) and IVSC (SEQ ID NO: 53).

In one embodiment, the hydrogel is stable in aqueous solution at ambienttemperature for a period of at least 7 days, preferably at least 2 to 4weeks, more preferably at least 1 to 6 months.

In one embodiment, at least 5%, preferably at least 10%, more preferablyat least 15%, more preferably at least 20%, more preferably at least25%, more preferably at least 30%, more preferably at least 35%, morepreferably at least 40%, more preferably at least 45%, even morepreferably at least 50% of said plurality of amphiphilic peptides and/orpeptoids are chemically cross-linked.

In one embodiment, at least 60% of said plurality of amphiphilicpeptides and/or peptoids are chemically cross-linked. In one embodiment,the hydrogel is characterized by a storage modulus G′ to loss modulus G″ratio that is greater than 2.

In one embodiment, the hydrogel is characterized by a storage modulus G′from 100 Pa to 400, 00 Pa, preferably 500 Pa to 400, 00 Pa, even morepreferably 1000 Pa to 400, 00 Pa, at a frequency in the range of from0.02 Hz to 16 Hz.

In one embodiment, the hydrogel has a higher mechanical strength thancollagen or its hydrolyzed form (gelatine).

In one embodiment, the hydrogel has an elasticity defined as % strain atlinear viscoelasticity (LVE) limit above 0.01% strain, preferably above0.5% strain, more preferably above 1% strain, more preferably above 2%strain.

In one embodiment, the hydrogel further comprises a non-peptidicpolymer.

In one embodiment, the hydrogel further comprises at least one of amicroorganism, a cell, a virus particle, a peptide, a peptoid, aprotein, a nucleic acid, an oligosaccharide, a polysaccharide, avitamin, an inorganic molecule, a nano- or microparticle, a syntheticpolymer, a small organic molecule, a cosmetic agent or apharmaceutically active compound.

In one embodiment, said at least one of a microorganism, a cell, a virusparticle, a peptide, a peptoid, a protein, a nucleic acid, anoligosaccharide, a polysaccharide, a vitamin, an inorganic molecule, anano- or microparticle, a synthetic polymer, a small organic molecule, acosmetic agent or a pharmaceutically active compound is entrapped bysaid three-dimensional macromolecular nanofibrous networks.

In one embodiment, said at least one of a microorganism, a cell, a virusparticle, a peptide, a peptoid, a protein, a nucleic acid, anoligosaccharide, a polysaccharide, a vitamin, an inorganic molecule, anano- or microparticle, a synthetic polymer, a small organic molecule, acosmetic agent or a pharmaceutically active compound is coupled to saidamphiphilic peptides and/or peptoids, preferably via a disulfide bridge.

In one embodiment, said at least one of a microorganism, a cell, a virusparticle, a peptide, a peptoid, a protein, a nucleic acid, anoligosaccharide, a polysaccharide, a vitamin, an inorganic molecule, anano- or microparticle, a synthetic polymer, a small organic molecule, acosmetic agent or a pharmaceutically active compound is coupled to saidnon-peptidic polymer. In one embodiment, said pharmaceutically activecompound is selected from the group consisting of haemostatic agents,antibiotics, anti-microbial agents, anti-fungal agents,anti-inflammatory agents, analgesics, anti-coagulants, antibodies,antigens, growth factors and cytokines. In one embodiment, said nano- ormicroparticle is a metal nano- or microparticle, preferably a gold nano-or microparticle.

In one embodiment, said peptide comprises a signal sequence, wherein,preferably, said peptide is coupled to said amphiphilic peptides and/orpeptoids via a disulfide bridge.

In one embodiment, said signal sequence comprises an adhesion or growthsignal, such as an integrin binding sequence (e.g. CRGD).

In one embodiment, said hydrogel is provided in an injectable form andgels in situ. The objects of the present invention are also solved by amethod of preparing a hydrogel, preferably a hydrogel according to thepresent invention, the method comprising the step of dissolvingamphiphilic peptides and/or peptoids, as defined above in connectionwith the hydrogel according to the present invention, in an aqueoussolution, wherein said aqueous solution comprises an oxidizing agent orwherein said method further comprises the step of exposing theready-made hydrogel to a solution of an oxidizing agent.

In one embodiment, said oxidizing agent is H₂O₂, wherein, preferably,H₂O₂ is used at a concentration from 0.02 to 0.1% (w/w), preferably 0.04to 0.08% (w/w), more preferably 0.05 to 0.07% (w/w).

In one embodiment, said amphiphilic peptides and/or peptoids aredissolved at a concentration from 0.01 μg/ml to 50 mg/ml, preferably ata concentration from 1 mg/ml to 25 mg/ml, more preferably at aconcentration from 1 mg/ml to 15 mg/ml, even more preferably at aconcentration from 5 mg/ml to 12 mg/ml.

In one embodiment, the dissolved amphiphilic peptides and/or peptoids inaqueous solution are further exposed to a temperature in the range offrom 20° C. to 90° C., preferably 20° C. to 70 C, more preferably 20° C.to 40° C.

In one embodiment, the dissolved amphiphilic peptides and/or peptoids inaqueous solution are exposed to said temperature for at least 1 hour,preferably at least 2 hours, more preferably at least 4 hours, morepreferably at least 6 hours, more preferably at least 8 hours, morepreferably at least 10 hours, more preferably at least 12 hours, evenmore preferably at least 24 hours.

In one embodiment, the method further comprises the step of exposing theready-made hydrogel to an aqueous solution not comprising said oxidizingagent, wherein, if said method comprises the step of exposing theready-made hydrogel to a solution of said oxidizing agent, said step ofexposing the ready-made hydrogel to an aqueous solution not comprisingsaid oxidizing agent is performed after said step of exposing theready-made hydrogel to a solution of said oxidizing agent.

In one embodiment, said step of exposing the ready-made hydrogel to anaqueous solution not comprising said oxidizing agent is repeated atleast once.

In one embodiment, said step of exposing the ready-made hydrogel to anaqueous solution not comprising said oxidizing agent occurs for at least1 hour, preferably at least 2 hours, more preferably at least 4 hours,even more preferably at least 6 hours.

In one embodiment, said step of exposing the ready-made hydrogel to anaqueous solution not comprising said oxidizing agent occurs at atemperature in the range of from 30° C. to 45° C., preferably 35° C. to40° C.

The step of exposing the ready-made hydrogel to an aqueous solution notcomprising said oxidizing agent is used to remove unreacted oxidizingagent and/or residual acid from solid-phase peptide synthesis. In oneembodiment, more than 90%, preferably more than 95%, more preferablymore than 97%, even more preferably more than 99% of the unreactedoxidizing agent (e.g. H₂O₂) are removed.

In one embodiment, said aqueous solution not comprising said oxidizingagent is water or a buffered aqueous solution (e.g. PBS).

In one embodiment, said aqueous solution not comprising said oxidizingagent is a cell culture medium. In one embodiment, the method furthercomprises at least one of the steps of:

-   -   adding at least one of a microorganism, a cell, a virus        particle, a peptide, a peptoid, a protein, a nucleic acid, an        oligosaccharide, a polysaccharide, a vitamin, an inorganic        molecule, a nano- or microparticle, a synthetic polymer, a small        organic molecule, a cosmetic agent or a pharmaceutically active        compound;    -   adding at least one non-peptidic polymer;    -   adding at least one gelation enhancer; —adding at least one        buffer, preferably at least one physiologically acceptable        buffer.

In one embodiment, said gelation enhancer is a salt or a solution of asalt. The objects of the present invention are also solved by a hydrogelprepared by the method according to the present invention.

The objects of the present invention are also solved by the use of ahydrogel according to the present invention as a cell culture substrate,preferably a cell culture substrate for 3-D cell culture.

The objects of the present invention are also solved by the use of ahydrogel according to the present invention as a device for drug or genedelivery, preferably for sustained or controlled release drug delivery,or as a wound dressing or as an implant or as an injectable agent thatgels in situ.

The objects of the present invention are also solved by a cell culturesubstrate, preferably a cell culture substrate for 3-D cell culture,comprising a hydrogel according to the present invention.

The objects of the present invention are also solved by a device fordrug or gene delivery, preferably sustained or controlled release drugdelivery, comprising a hydrogel according to the present invention.

The objects of the present invention are also solved by an implant orinjectable agent or wound dressing comprising a hydrogel according tothe present invention.

The objects of the present invention are also solved by a pharmaceuticalor cosmetic composition comprising a hydrogel according to the presentinvention.

In one embodiment, the pharmaceutical or cosmetic composition isprovided in the form of a topical gel or cream, a spray, a powder, or asheet, patch or membrane.

In one embodiment, the pharmaceutical or cosmetic composition isprovided in the form of an injectable solution. In one embodiment, thepharmaceutical or cosmetic composition further comprises apharmaceutically active compound.

In one embodiment, the pharmaceutical or cosmetic composition furthercomprises a pharmaceutically acceptable carrier.

The objects of the present invention are also solved by a hydrogelaccording to the present invention for use in regenerative medicine orfor use in tissue engineering and tissue regeneration, e.g. regenerationof adipose and cartilage tissue.

The objects of the present invention are also solved by a hydrogelaccording to the present invention for use in the treatment of wounds.

The objects of the present invention are also solved by a hydrogelaccording to the present invention for use in the treatment ofdegenerative diseases of the skeletal system, e.g. degenerative discdisease, or urinary incontinence.

The objects of the present invention are also solved by a hydrogelaccording to the present invention for cosmetic use.

The objects of the present invention are also solved by an electronicdevice comprising a hydrogel according to the present invention.

In one embodiment, the electronic device is selected from a fuel cell, asolar cell, an electronic cell or a biosensing device.

The objects of the present invention are also solved by a method oftissue regeneration or tissue replacement comprising the steps:

-   -   a) providing a hydrogel according to the present invention;    -   b) exposing said hydrogel to cells which are to form regenerated        tissue;    -   c) allowing said cells to grow on or in said hydrogel.

In one embodiment, the method is performed in vitro or in vivo or exvivo.

In one embodiment, the method is performed in vivo, wherein, in step a),said hydrogel is provided at a place in the body of a patient wheretissue regeneration or tissue replacement is intended. In oneembodiment, said tissue is selected from the group comprising skintissue, nucleus pulposus in the intervertebral disc, cartilage tissue,synovial fluid and submucosal connective tissue in the bladder neck. Inone embodiment, said step a) is performed by injecting said hydrogel ora solution of amphiphilic peptides and/or peptoids as defined above inconnection with the hydrogel according to the present invention at aplace in the body of a patient where tissue regeneration or tissuereplacement is intended. In one embodiment, said step a) furthercomprises the co-injection of a gelation enhancer, preferably of asolution of a salt, and/or the co-injection of an oxidizing agent.

In one embodiment, the method is performed ex vivo, wherein, in step a)or b), cells from a patient or from a donor are mixed with saidhydrogel, and the resulting mixture is provided at a place in the bodyof a patient where tissue regeneration or tissue replacement isintended.

In one embodiment, said hydrogel comprises one or more bioactivetherapeutics that stimulate regenerative processes and/or modulate theimmune response.

Other aspects and features of the present invention will become apparentto those skilled in the art upon review of the following description ofspecific embodiments of the invention in conjunction with theaccompanying FIGS. 1K-M and 17 to 40.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the figures, wherein:

FIGS. 1A to 1M represent a sorted list of some exemplary peptidescapable of forming hydrogels. These peptides are embodiments in whichthe entire peptide consists of a single linear amphiphilic sequence.Peptides which are forming hydrogels are named with a short code, buttheir individual sequence is disclosed. The peptides of these examplesconsist of a sequence of natural amino acids containing 3 to 7 aminoacids. The N-terminus is acetylated which removes the charge that wouldotherwise restrain the amphiphilic character of the peptides.

FIG. 2 depicts gelation pictures for peptide based hydro gels at lowestconcentrations.

FIG. 3 depicts gelation pictures for Ac-AS-6 (Ac-AIVAGS) (L) atconcentrations of 5 mg/ml, 10 mg/ml, 15 mg/ml.

FIG. 4 depicts a hypothesis of self-assembly from peptide monomers tosupramolecular network of condensed fibers. (A) Assembly is believed toinitiate with antiparallel pairing of two peptide monomers by changingto a-helical conformations. Subsequently, peptide pairs assemble tofibers and nanostructures. Condensation of peptide fibers to fiberaggregates results in hydrogel formation.

FIGS. 5A-C depict environmental scanning electron microscopy (ESEM)images of hydrogels of Ac-LD6 (Ac-LIVAGD) (L) (10 mg/ml), where FIG. 5A,FIG. 5B and FIG. 5C are images obtained at magnification of 260×, 1000×,2000×, 2400×, 4000× at a temperature of 4° C. with HV at 10 KV. Theimages indicate the formation of fibrous structures.

FIGS. 6A-D show field emission scanning electron microscopy (FESEM)images of hydrogels of Ac-LD6 (Ac-LIVAGD) (L) (15 mg/ml), where FIGS.6A-D are images obtained at magnifications of 6000×, 45000×, 45000× and40000× with HV at 10 KV.

FIGS. 7A-B depict field emission scanning electron microscopy (FESEM)images of Ac-AD6 (Ac-AIVAGD) (D) hydrogels (20 mg/ml) at a magnificationof 50× (FIG. 7A) and 20000× (FIG. 7B) at 12 KV.

FIGS. 8A-B show field emission scanning electron microscopy (FESEM)images of hydrogels of Ac-AD6 (Ac-AIVAGD) (D) (20 mg/ml) obtained at120× (FIG. 8A), and 450× (FIG. 8B).

FIG. 9A shows the morphology and structure evaluation of the peptidescaffolds as determined by field emission scanning electron microscopy(a-f). (a) A honeycomb porous structure is observed followinglyophilization of 20 mg/mL Ac-AD₆ (Ac-AIVAGD) (D) hydrogel. The poresare bounded by membranes of condensed fibers as shown in close-up viewsof 15 mg/mL (b) and 20 mg/mL (c) Ac-ID₃ (Ac-IVD) (L) hydrogels. Furthermagnification of 20 mg/mL Ac-AD₆ (L) hydrogel revealed single fibers (d,e). At lower concentrations, 0.1 mg/mL Ac-LD₆ (Ac-LIVAGD) (L),nanostructures are observed (f).

FIG. 9B shows an image obtained at a magnification of 1000×, HV of 12KV, FIG. 9C obtained at a magnification of 2500×, HV of 12 KV, FIG. 9Dobtained at a magnification of 4000×, HV of 10 KV, FIG. 9E obtained at amagnification of 35000×, HV of 10 KV, FIG. 9F at a magnification of80000×, HV of 5 KV, FIG. 9G obtained at a magnification of 120000×, HVof 10 KV, and FIG. 9H at a magnification of 200000×, HV of 10 KV.

FIG. 10A shows Far-UV CD spectra demonstrating that with increasingconcentration there is the transition of Ac-LD₆ (Ac-LIVAGD) peptideconformation from random coil (below threshold concentration) toα-helical (222 and 208 nm peaks) and further β-type (negative band at218 nm) structures. Heating the samples to facilitate gelation increasedthe β type aggregation. FIG. 10B Below threshold concentration, therandom coil conformations of 0.2 mg/mL Ac-LD₆ were reversibly affectedby step-wise temperature increases (solid lines) from 25° C. to 90° C.and cooling (dotted lines). FIGS. 10C and 10D Above the thresholdconcentration in 1 mg/mL Ac-LD₆ gel, stepwise temperature increases FIG.10C stabilized the β-type structures irreversibly, such that subsequentcooling FIG. 10D did not alter the CD spectra. FIG. 10E Far-UV CDspectra of AcID₃ (Ac-IVD) at different concentrations. All curves weredone at 25° C.

FIGS. 11A and 11B show rheological measurements. The high mechanicalstrengths of different peptide hydrogels at 20 mg/mL concentration wasdetermined by measuring storage moduli (G′) as a function of angularfrequency under 0.1% strain, at 25° C. and 50° C. respectively. The gelsdemonstrate good thermal stability compared to gelatin, which liquifiedat 50° C. (hence excluded in 4B). FIG. 11C Mechanical strength is afunction of concentration, as determined from oscillatory frequencysweep studies using Ac-LD₆ (Ac-LIVAGD) (L) under 0.1% strain at 25° C.FIG. 11D Increasing salt concentration (NaCl) decreases G′, reducing therigidity of 10 mg/mL Ac-LD₆ (L) hydrogels, demonstrating the tunabilityand reversibility of gelation.

FIGS. 12A-D show further examples of a rheology measurements for peptidebased hydrogels. FIG. 12A and FIG. 12B depict oscillatory amplitudesweep studies at temperatures of 25° C. and 50° C. for Ac-AD6(Ac-AIVAGD) (L) and Ac-AD6 (D) at a concentration of 20 mg/ml with aconstant frequency of [1rad-s] and a gap of 0.8 mm. The graphs indicatethe plot of moduli [Pa] versus strain (%) at temperatures of 25° C. and50° C. The linear viscoelastic range was observed at 0.07% to 0.2 strain% at temperatures of 25° C. and 50° C. FIG. 12C and FIG. 12D depictoscillatory frequency sweep Studies at temperatures of 25° C. and 50° C.for Ac-AD6(L) and Ac-AD6(D) at a concentration of 20 mg/ml with varyingfrequency ranges from 0.1 to 100 [Rad/s] with a constant strain [%] of0.1% linear viscoelastic range and a gap of 0.8 mm.

FIG. 13 shows a further example of a rheology measurement for peptidebased hydrogels. Depicted is a frequency sweep study of a UVcross-linked peptide at a temperature of 25° C. with 0.1% strain.

FIG. 14 depicts rheology measurements for gelatin-1890 (type A, porcineskin). This figure shows moduli data obtained at 25° C. when applyingdifferent frequencies.

FIGS. 15A-D illustrate the biocompatibility of peptide-based hydrogelsof the invention using further cell lines. FIG. 15A shows a microscopyimage of human primary renal tubule cells (HPRTC) after 72 hours afterseeding on a hydrogel of Ac-LD₆ (Ac-LIVAGD) (L) in DMEM medium, grown atoptimum conditions. FIG. 15B shows microscopy images of human primaryrenal tubule cells (HPRTC) after 72 hrs after seeding on tissue cultureplastic, grown at optimum conditions. FIG. 15C shows microscopy imagesof human umbilical vein endothelial cells (HUVEC) after 72 hrs afterseeding on gels of Ac-LD₆ (L) in DMEM medium, grown at optimumconditions. FIG. 15D shows microscopy images of human umbilical veinendothelial cells (HUVEC) after 72 hrs after seeding on tissue cultureplastic, grown at optimum conditions.

FIGS. 16A-B are further illustrations on the viability of cells inpresence of a hydrogel of the invention. Human fibroblast cells werecultured in the presence (FIG. 16A) and absence (FIG. 16B) of Ac-LD₆(Ac-LIVAGD) (L) (5 mg/ml). Fluorescein isothiocyanate (FITC) stainedcells (left panels), Texas red stained cells (center panels) and cellsstained with both FITC and Texas red (right panels) are shown.

Embodiments of the invention will now be described by way of examplewith reference to the following figures, in which:

FIG. 17 shows the crosslinking strategy of the present invention using(A) disulfide bridges between two thiol-containing amino acids (here:two cysteines) which introduce (B) chemical intra- and inter-fibercrosslinks among LK₆C peptide fibers. The thiol groups furtherfacilitate the conjugation of bioactive agents, such as the integrinbinding sequence CRGD (C).

FIG. 18 at A illustrates the determination of a suitable gelationconcentration with the LK₆ control peptide sequence. A workingconcentration was established that was suitably low so as to save onmaterial during testing but yet, capable of forming gels strong enoughto be manipulated. LK₆ gels were therefore casted at differentconcentrations and their stiffness was measured (average±s.d. oftriplicates). As expected, G′ values increased with peptideconcentration and it was determined that a working concentration of 12mM afforded gels with sufficient mechanical integrity to withstandhandling. FIG. 18 at B shows that gels can be formed with 12 mM of LK₆C(˜10 mg/mL after accounting for an amino acid content of 89.4%) and thatgel formation is compatible with various aqueous media.

FIG. 19 shows the kinetics of disulfide formation when LK₆C wassubjected to oxidation by air, as compared to H₂O₂-assistedoxidation+/−HRP (average±s.d. of duplicates). For oxidation by air, LK₆Cdissolved in water at 12 mM was left in capped micro-centrifuge tubes atroom temperature. As can be seen, kinetics of disulfide formation wassluggish and ˜80% of thiols still remained after 5 days, as determinedusing an Ellman's assay. H₂O₂-assisted oxidation was much more efficientin the formation of disulfide bridges. Interestingly, horse-radishperoxidase (HRP, 0.6 U/mL), an enzyme commonly used with H₂O₂ to boostoxidation efficiency, did not significantly increase the rate ofdisulfide formation.

FIG. 20 shows representative UPLC chromatograms where the area under thepeaks were used to follow the rate of disulfide formation inH₂O₂-assisted oxidation. 12 mM LK₆C dissolved in water containing 0.06%H₂O₂ was used, and the disulfide peak at ˜3.6 min gradually increased.Only ˜50% of thiols remained after 1 day. The R² value of the thiolcalibration curve was 1.000.

FIG. 21 shows mass spectra corresponding to the a) LK₆C monomer-thioland b) (LK₆C)₂ dimer-disulfide peaks in the UPLC elution profile. Inboth cases, the expected masses were detected and verified theassignment of peaks.

FIG. 22 shows the effect of the H₂O₂ concentration on the rate ofdisulfide formation. LK₆C was incubated with different concentrations ofH₂O₂ for eight hours at 25° C. before UPLC analysis. The area under thethiol peaks were then normalised to that at 0 hour to give the % ofthiol remaining. Before that, a calibration curve had been generatedwith pure LK₆C as standard (R²=0.999). As expected, a higherconcentration of H₂O₂ increased the kinetics of disulfide formation.However, as a compromise between oxidation efficiency andH₂O₂-associated toxicity, 0.06% H₂O₂ was chosen for subsequentexperiments.

FIG. 23 illustrates two different methods to effect cross-linking: 1)cast-and-soak: gel is first formed in water and then soaked in a H₂O₂solution for the desired amount of time. 2) in situ oxidation: LK₆Cpeptide powder is dissolved directly in water containing H₂O₂ to formthe gel.

FIG. 24 shows that oxidation dramatically improved the ability of thegels to retain their shapes, as seen in panels A to D: (A) LK₆C gelcasted at the start of experiment which was either (B) not oxidised andsoaked in water for 24 hours, or (C) oxidised for two hours and soakedin water for 96 hours. (D) The control peptide sequence, LK₆, did notsurvive the 24-hour water soak.

FIG. 25 and FIG. 26 show that the hydrogel was uniformly oxidised in thecast-and-soak method. In the cast-and-soak method, LK₆C was dissolved inwater, casted overnight in a ring mould before being soaked in asolution containing H₂O₂ for the desired amount of time (FIG. 25A). Toinvestigate if oxidation was uniform throughout the bulk or enrichedonly in the surface layers, the gel was removed from the H₂O₂ solutionafter two hours and carefully separated into its surface (top, bottomand circumference) and core fractions using a surgical blade (FIG. 25Band FIG. 26A). The amount of H₂O₂ in the respective fractions wasquantified using the PeroXOquant H₂O₂ assay kit (Pierce, Ill., USA),according to the manufacture's recommendations (average±s.d. oftriplicates). Samples were always diluted such that absorbance readingsfell within the linear portion (R²=0.999) of the calibration curveobtained with H₂O₂ standards (FIG. 25C). The gel fractions were alsoanalysed using UPLC (FIG. 26B; average±s.d. of duplicates). To normalisefor any differences in fiber density between the surface and corelayers, the area ratio between the disulfide and thiol (D/T) peaks wastaken to indicate the extent of oxidation that had occurred in thefractions. As can be seen, the amount of H₂O₂ detected in both thefractions was comparable, suggesting that the diffusion of small H₂O₂molecules was extremely efficient. Compared to without oxidation, theD/T ratio of both layers was, as expected, significantly elevated afteroxidation. However, the D/T ratio of the surface and core layer wasinsignificantly different after oxidation, indicating that the gel wasuniformly oxidised in the cast-and-soak method.

FIG. 27 illustrates the rheological properties and the effects ofoxidation on the microstructure and ability of LK₆C gels to retentiontheir shape. The (A) stiffness and (B) elasticity of LK₆C gels weremeasured after various oxidation regimes (average±s.d. of triplicates).Generally, elasticity increased whereas stiffness was either maintainedor increased after the introduction of S—S bonds.

FIG. 28 illustrates the rheological properties of oxidized hydrogels. 12mM LK₆C gels were subjected to various oxidation regimes with 0.06% H₂O₂and various durations of water soak before their (A) stiffness and (B)elasticity were measured (average±s.d. of triplicates). Withoutoxidation, LK₆C gels could maintain their stiffness after two hours ofwater soak but broke down and lost their stiffness after 24 hours ofwater soak. On the other hand, gels subjected to 2 or 24 hours ofcast-and-soak oxidation, or 22 hours of in situ oxidation maintainedtheir shapes and in fact, became stiffer after up to four days of watersoak. Presumably, the introduction of chemical S—S bonds was importantin helping to keep the peptide fibers together and increase the abilityof the gels to maintain their shape and stiffness. Compared to gelswithout oxidation, the increase in elasticity of the oxidised gels wasalso maintained after up to four days of water soak.

FIG. 29 shows the effects of concentration and LK6-doping on therheological properties of LK₆C. LK₆C was dissolved in water at 12 or13.5 mM and casted overnight in a ring mould. The (A) stiffness and (B)elasticity of the gels were then measured (average±s.d. of triplicates).Increasing the concentration of LK₆C increased the gel stiffnesssignificantly but only resulted in a modest increase in elasticity.(C-D) LK₆C was also mixed with varying amount of LK6 to a finalconcentration of 12 mM in water and casted overnight in a ring mould.The “%” values presented above refer to the proportion of LK₆C in theformulation. The gels were then soaked in a 0.06% H₂O₂ solution for twohours (i.e., cast-and-soak) before their (C) stiffness and (D)elasticity were measured (average±s.d. of triplicates). As can be seen,pure LK₆C (100%), pure LK6 (0%) and LK₆C/LK6 75/25 (75%) gels wererelatively stiff. Gels containing ≤50% LK₆C, however, broke downbefore/during handling, resulting in low G′ values. These suggest thatthe window to dope LK₆C with LK₆ (at a final concentration of 12 mM)while still maintaining gel stiffness, lies between 75-50% of LK₆C. Gelscontaining oxidised LK₆C were more elastic than pure LK₆ gels. However,the difference in elasticity between gels containing 75% and 100% ofLK₆C was only modest.

FIG. 30 depicts FESEM images showing that the fibrous microstructure ofLK₆C was maintained after oxidation. Freeze-dried gels were depositedonto carbon tapes, sputtered with platinum and observed under aJSM-7400F electron microscope (Jeol, Tokyo, Japan).

FIG. 31 illustrates the purification of hydro gels before their use incell culture. LK₆C was dissolved in 200 μL of water containing 0.06%H₂O₂ and casted directly into a 48-well plate for 22 hours at 25° C. 1mL of water was then added on top of the gel and replaced at regulartime intervals to leach out (A) unreacted H₂O₂ and (B) residual H⁺ fromsolid-phase peptide synthesis (average±s.d. of duplicates at least). Theamount of H₂O₂ in the supernatant was quantified as before and plottedwith respect to time. The total amount of H₂O₂ at the start ofexperiment was obtained by: sum of all removed H₂O₂ in thesupernatants+whatever amount of H₂O₂ remaining in the gel at the end ofexperiment. The amount of H⁺ removed was determined by measuring the pHof the supernatant with respect to control wells with no gel (onlywater). Since pH is proportional to the negative logarithm of theactivity of H⁺ ions, the amount of H⁺ removed from the gel can becalculated with respect to the control wells, which account for naturalair acidification. Experiment was stopped when pH of supernatant matchedthat of the control wells and cumulative activity of H⁺ removed wasplotted as a function of time. As can be seen, >96% of H₂O₂ was removedafter 4 hrs and >99% after 7 hrs. Similarly, >85% of residual acid wasremoved after 8 hrs. We however note that as the purification efficiencyis diffusion controlled, the plots above are only valid for the reportedfrequency of water change. A more frequent regime of water change orusing a buffered media (e.g., growth media or PBS) instead of water isexpected to give even better results.

FIG. 32 illustrates the gradual and tunable release kinetics of thehydro gels according to the present invention. LK₆C containing 1 mg/mLof dextran-dye (10 kDa) was first casted in 48-well plates and incubatedovernight. The hydrogel was either not oxidized or oxidized for 2 hrsbefore water was introduced at room temperature. Water was thenextracted to assay for dextran release from the hydrogel (R² ofcalibration curve was 0.999). The release profile showed no burstrelease, but a gradual release up till 28 days. The release rate of theoxidized hydrogel was lower, presumable due to its greater stability inwater.

FIG. 33 shows the chemical conjugation of CRGD onto LK₆C fibers viacysteine-mediated disulfide bonds. CRGD was mixed with LK₆C in thepresence of 0.06% H₂O₂ and casted in a ring mould at 25° C. for 22hours. (A) UPLC later revealed that there were three main peaks in thechromatogram which could be assigned, based on their respective MS, to:(B) unreacted LK₆C monomers, (C) (LK₆Ch dimers and (D) disulfide-linkedLK₆C−CRGD conjugates. All peaks have been colour-coded to facilitateinterpretation. Earlier experiments also determined that unreacted CRGDmonomers or (CRGD)₂ dimers eluted at ˜1.1 min (data not shown), both ofwhich were not observed here (see magnified inset of A). Theintegrin-recognition motif, CRGD was therefore successfully conjugatedonto LK₆C fibers. The conjugation of the CRGD motif is only intended toillustrate the versatility of this platform and the simplicity ofreaction conditions. Future conjugations need not be limited to thissequence.

FIG. 34 shows the effects of 3D cell culture and the incorporation ofRGD on the viability and spreading of HepG2 cells. (A) En face views ofviable cells stained with calcein cultured on regular 2D surfaces forfour days, compared to cells in LK₆C+/−RGD gel culture. All images,except for the 2D culture, were mergers of several z-stacks. (B) Cellswere viable, as evident from the positive calcein signals, after fourdays of culture on the RGD functionalised, crosslinked and purified LK₆Cgels. 3D distribution was also achieved, as con-31 firmed bycross-sectional slices which revealed multi-layered cell growth. Thepresence of RGD increased the viability of cells, as visually suggestedin (A) and corroborated by (C) the MTT assay. (D) In the case of HepG2cells, LK₆C gel culture in the presence or absence of RGD hadinsignificant effects (p>0.05) on cell spreading compared to those inregular 2D culture. (C-D) Average±s.d. of triplicates at least.

FIG. 35 illustrates the 3D primary cell culture and their cell-spreadarea. (A) Fibroblasts isolated from the cornea/sclera of New ZealandWhite rabbits were maintained in regular completed DMEM and grown in 2Dculture or purified LK₆C gels+/−various concentrations of CRGD. Cellswere incubated for four days before they were stained with calcein AMfor confocal imaging. Images from gel cultures were mergers of severalz-stacks. As observed, viable fibroblasts were seen in all cases. In thecase of 2D culture, it was repeatedly observed that the cells wereheterogeneously distributed throughout the well area, i.e., the middleof the well was sparsely populated while the edges were confluent. Cellsin gel culture, on the contrary, were evenly distributed across the gel.We note that seeding of cells was done on the same day using the samemethods and most probably did not account for the in homogeneousdistribution of cells. (B) Images taken at a higher magnification werethen analysed using the ImageJ software to quantify the averagecell-spread area of the fibroblasts (Average±s.d. of triplicates atleast). The rabbit fibroblasts spreaded more in 2D culture compared tothose in the various gel cultures (ANOV A, p<0.05). There were nosignificant differences in terms of cell-spread area between gels withor without CRGD in them.

FIG. 36 shows confocal images (40× magnification) of HepG2 cells eithercultured on LK₆C/CRGD gels or on regular 2D wells. In the former, theimage presented was a merger of several z-stacks. Cells were stainedwith calcein AM and Hoechst (Invitrogen, Singapore) prior to confocalobservations. The images from at least two independent locations werethen processed with the ImageJ software to quantify the averagespreading area of cells.

FIG. 37 shows the rheological properties of gels of LK₆C (10 mg/mL) withor without CRGD (1 mg/mL) were casted overnight in the presence of 0.06%H₂O₂ before their (A) stiffness and (B) elasticity were quantified withoscillation rheometry (Average±s.d. of triplicates). Upon CRGDconjugation, the gel maintained their stiffness but became less elastic.

FIG. 38 shows that 3T3 murine fibroblasts cultured in 3D in LK₆C+CRGDhydrogels remained viable for at least 21 days. Cells were stained withcalcein AM prior to confocal microscopy.

FIG. 39 illustrates the surgical procedure used for testing LK₆C+CRGDhydrogels in the treatment of wounds. (1) Hair was removed, and the areafor surgical removal was marked. Both the epidermis and dermis wereremoved to simulate injury. The removed skin flap was killed by arepeated cycle of freezing and thawing. (2) Mouse with open wound. (3)LK₆C+CRGD hydrogels with or without 3T3 fibroblast cells were placedonto wound and the killed skin flap was sutured back to act as a bandagedressing. (4) Mouse after skin flap dressing was sutured back.

FIG. 40 shows that hydrogel treatment promotes vascularisation in wounds(see red arrows). Preliminary data obtained with H&E stained slidesrevealed that the gel+3T3+dressing group has the most significantre-epithelisation and regeneration of a thicker dermis (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have previously described short peptide sequences (3-7residues) capable of self-assembly into helical fibers that ultimatelyresult in hydrogel formation. While these peptides have good mechanicalproperties and can entrap large amount of water, they rely purely onphysical forces to keep the interwoven fiber network together.Consequently, some peptide sequences—one example of which is LIVAGK, orabbreviated to LK6—are typically used at rather high concentrations toensure the integrity and stability of the gel in aqueous solution. Thishas contributed significantly to cost.

The present invention as defined above offers significant improvementsin material properties such as stiffness, elasticity and resistance ofthe gel to degradation compared to previous disclosures-ultra-smallpeptide-based and peptide/polymer-based hydrogels. Reactive chemicalgroups are also introduced to the peptide fibers which allow the facilein-situ chemical conjugation of biological signals. Furthermore,compared to previous formulations, less material is now needed forgelation (e.g. 10 mg/ml LK₆C instead of 25 mg/ml for LK6), whichtranslates into significant cost savings and higher water contents. Thisclass of material is suitable for biomedical applications, for examplebut not limited to, three-dimensional cell culture, tissue engineering,sensing and drug and gene delivery.

The present invention is based on the introduction of chemical bonds tocrosslink the peptide fibers, resulting in gels with increasedstiffness, elasticity and resistance to degradation. According to thepresent invention chemical crosslinks are introduced by the modificationof the peptide sequence with a cysteine residue at the C-terminus, e.g.,LIVAGKC (or LK₆C). This way, the pure amino acid composition of the gelis still conserved, which makes it, in general, a biocompatiblematerial. Three-dimensional gels have been casted using thesecross-linkable peptides in water or cell culture medium, such as growthmedium completed with FBS/Penicillin/Streptomycin, and cargoes have beenencapsulated within the matrix. Crucially, on top of physical selfassembly forces, inter- and intra-fiber chemical bonds (in particular,disulfide bonds between cysteine or homocysteine residues which each hasa thiol group) are now introduced. These chemical bonds can beencouraged in the presence of an oxidising agent like hydrogen peroxide(H₂O₂). Alternatively, chemical bond (e.g. disulfide) formation can alsobe encouraged in the oxidative blood environment when used in vivo. Theformation of crosslinks significantly increases the elasticity of thegel due to the presence of additional chemical bonds. Another advantageof introducing crosslinks includes the increased resistance of the gelto degradation due to stronger interactions within and between fibers.This also means that less material is needed to obtain gels withmechanical properties comparable or even superior to earlierformulations, which in turn translates into significant cost savings andhigher water contents. The present invention also allows the tuning ofthe physical properties of the hydrogels (stiffness, elasticity etc.) bymodifying the oxidation cross-linking regime. The presence of reactivechemical groups also enables the functionalization of the gel withcargos or adhesive signals (e.g., CRGD) that further increase thebiocompatibility of the gel. Therefore, cargos can now be covalentlyattached to the gel matrix besides simple physical encapsulation. Theconjugation procedure can furthermore be conveniently done in situ,while the gel is being formed. According to the present invention, onlymild conditions for the hydrogen peroxide-assisted oxidation process areused. Importantly, the use of horse-radishperoxidase (HRP) prescribed inmany other oxidation protocols can be avoided. This further reduces costand facilitates regulatory approval in the future. Moreover, theinventors have devised a method to remove most of the unreacted hydrogenperoxide (>99%) and residual acid from solid-phase peptide synthesisbefore the introduction of e.g. cells onto the gels. The release profileof cargoes encapsulated within the gel can also be tuned by modifyingthe oxidation strategy.

The hydrogels according to the present invention are non-allergenic andnon-toxic. The inventors also showed the biocompatibility of thehydrogels by successfully culturing several cell types on purified andcrosslinked LK₆C−CRGD gels. They also managed to show the 3Ddistribution of cells within LK₆C−CRGD hydrogels. More particularly,cells were seeded on the gel and stained with the fluorescent live cellmarker calcein. By obtaining a vertical cross sectional slice of thehydrogel and directly imaging the penetration depth profile of thecells, they observed multi-layered cell growth. This confirmed theinfiltration of cells into the gel and convincingly demonstrated the 3Dcell growth environment.

Potential applications of the hydrogels according to the presentinvention include:

-   -   (Injectable) application for tissue engineering, particularly in        orthopaedic and aesthetic surgery applications. H₂O₂ may even be        avoided altogether as the overall redox potential of the blood        is oxidative (while the intracellular overall redox potential is        reductive). Thus, disulfide bonds will be naturally formed and        in the process and, thus, increase the long-term stability of        gel, which is already an improvement over current formulations.    -   3D cell culture substrate. Adhesion or growth signals can be        covalently attached to increase biocompatibility. Such 3D cell        culture substrate may, for example, be used as 3D cancer model.    -   Skin Grafting. Using e.g. an LK₆C-based hydrogel as a scaffold,        first fibroblasts are embedded within the bulk of the gel,        followed by seeding of keratinocytes onto the surface of the gel        to mimic the dermis and epidermis, respectively, of the human        skin. This artificial skin layer can then be explored for        grafting applications.    -   Scaffold for corneal endothelial transplantation. The        endothelium of a human cornea is a monolayer and is critical to        the maintenance of vision. Handling of the endothelium is        therefore technically challenging and can influence the success        of a corneal endothelial transplant. Hydrogels according to the        present invention, such as those based on LK₆C, could be used as        a matrix support, on which corneal endothelial cells are        cultured, and which can then be used as an endothelium        replacement for transplants.    -   Cargo delivery. Drugs and nanoparticles, e.g., EI/DNA complexes        can be encapsulated for gradual release. Physical concentration        of cargo in the vicinity of cells. Less cargo may be needed to        achieve similar effects (compared to dilution of particles by        dripping directly into growth medium), reducing on cost and        toxicity.    -   Surface adhesion of cargoes, e.g., DNA solution can first be        layered on top of gel. Electrostatic interactions can hold DNA        on surface of gel before DNA solution removed and cells cultured        over gel. In this case, localised transfection is achieved.    -   Attachment of particles, e.g., gold particles for application in        sensing or other biodevices.    -   Wound treatment. Advantages of using hydro gels according to the        present invention for the treatment of wounds include: the        hydrogel keeps the wound moist; it provides a scaffold for the        regenerating skin layers; therapeutics can be loaded into the        gel to aid recovery.

Disclosed herein is a novel class of hydrogels comprisinghydrogel-forming peptides/peptoids derived from inter alia natural aminoacids. These peptides/peptoids are small amphiphilic peptides with ahydrophobic portion of aliphatic amino acids and, preferably, one or twopolar amino acids. The peptides/peptoids (typically 3-7-mers) aretypically in the L- or D-form and can self assemble into supramolecularfibers which are organized into mesh-like structures. The hydrogels aregenerally characterized by a remarkable rigidity and are biocompatibleand non-toxic. Depending on the peptide/peptoid sequence these hydrogelscan show thermoresponsive or thixotropic character. By selecting thepeptide assembling conditions the thickness and length of the fibers canbe controlled. The rigid hydro gels can be used for cultivation of avariety of primary human cells, providing peptide scaffolds that can beuseful in the repair and replacement of various tissues. Also disclosedis the procedure of preparing these hydrogels. Disclosed is further theuse of respective hydro gels in applications such as cell culture,tissue engineering, plastic surgery, drug delivery, oral applications,cosmetics, packaging and the like as well as for technical applications,as for example for use in electronic devices which may include solar orfuel cells.

Also disclosed herein is an amphiphilic peptide and/or peptoid capableof forming a hydrogel, i.e. a polymer network in which water is thedispersion medium. The amphiphilic peptide and/or peptoid includes oneor more linear amphiphilic sequences, each having a polar and anon-polarportion. For sake of simplicity explanations are in the following to alarge extent focused on amphiphilic peptides and/or peptoids thatconsist of a single respective linear sequence. In these explanations arespective peptide and/or peptoid is denominated a “linear peptideand/or peptoid”. Respective explanations apply to any linear sequence,which may also be included in an amphiphilic peptide and/or peptoid witha plurality of these linear sequences. Each of these linear sequences isindividually selected. In some embodiments an amphiphilic peptide and/orpeptoid disclosed herein includes several linear amphiphilic sequences,each of them differing from any other of the linear amphiphilicsequences. In some embodiments an amphiphilic peptide and/or peptoiddisclosed herein includes several identical linear amphiphilicsequences. In one embodiment an amphiphilic peptide and/or peptoiddisclosed herein includes a plurality of linear amphiphilic sequences,each linear amphiphilic sequence being identical to each other linearamphiphilic sequence.

Also disclosed is a peptide and/or peptoid that includes o amphiphiliclinear sequences. The symbol o represents an integer selected in therange from 1 to about 25, such as from 1 to about 20, from 1 to about18, from 1 to about 15, from 1 to about 12, from 1 to about 10, from 1to about 8, from 1 to about 6, from 1 to about 5 from 1 to about 4 orfrom 1 to about 3.

In some embodiments these amphiphilic linear sequences are linked in aconsecutive manner, thereby defining a linear portion of the peptideand/or peptoid.

In some embodiments the peptide and/or peptoid has a backbone with oneor more branches. In such an embodiment these amphiphilic linearsequences may be included on different branches. As mentioned above,each of the o amphiphilic linear sequences is independently selected. Arespective amphiphilic linear sequence has a length of n aliphatic aminoacids. The symbol n represents an integer selected in the range from 3to about 18, such as from 3 to about 15, from 3 to about 14, from 3 toabout 13, from 3 to about 12, from 3 to about 11, from 3 to about 10,from 3 to about 9, from 3 to about 8 or from 3 to about 7, such as 3, 4,5, 6, 7, 8, 9 or 10 aliphatic amino acids.

In some embodiments an amphiphilic linear sequence of a peptide and/orpeptoid described herein is chiral, rendering the entire amphiphilicpeptide and/or peptoid chiral. A corresponding linear peptide and/orpeptoid, i.e. an embodiment that consists of a single respective linearsequence, is accordingly a chiral peptide or peptoid. A respectiveamphiphilic linear sequence may include any linear non-aromatic aminoacid. The term “amino acid” as used herein refers to an alpha-aminocarboxylic acid, i.e. a carboxylic acid with an amino group in theα-position. The respective amino group may be an —NH² group or an —NHR¹group. The moiety R¹ may be any aliphatic group, whether alkyl, alkenylor alkynyl, with a main chain that includes 1 to 5, to 10, to 15 or to20 carbon atoms. Examples of alkenyl radicals are straight chain orbranched hydrocarbon radicals which contain one or more double bonds.Alkenyl radicals generally contain about two to about twenty carbonatoms and one or more, for instance two, double bonds, such as about twoto about ten carbon atoms, and one double bond. Alkynyl radicalsnormally contain about two to about twenty carbon atoms and one or more,for example two, triple bonds, preferably such as two to ten carbonatoms, and one triple bond. Examples of alkynyl radicals arestraight-chain or branched hydrocarbon radicals which contain one ormore triple bonds. Examples of alkyl groups are methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, the n isomers ofthese radicals, isopropyl, isobutyl, isopentyl, sec-butyl, tert-butyl,neopentyl, 3, dimethylbutyl.

A peptoid is an oligo(N-alkyl) glycine that, similar to the side chainconnected to the a carbon atom (see below) of a peptide, at the amidenitrogen carries a moiety that is an aliphatic moiety. Accordingly, inembodiments where an —NHR¹ group (supra) is included in the amino acidand the a carbon atom is included in a —CH₂— group, the reaction productof coupling a plurality of such amino acids may be called a peptoid. Apeptoid can also be taken to differ from a peptide in that it carriesits side chain at the amide nitrogen rather than at the a carbon atom.Peptoids are typically resistant to proteases and other modifyingenzymes and can have a much higher cell permeability than peptides (seee.g. Kwon, Y.-U., and Kodadek, T., J. Am. Chem. Soc. (2007) 129,1508-1509).

The term “amino acid” includes compounds in which the carboxylic acidgroup is shielded by a protecting group in the form of an ester(including an ortho ester), a silyl ester, an amide, a hydrazide, anoxazole, an 1,3-oxazoline or a 5-oxo-1,3, oxazolidine. The term “aminoacid” also includes compounds in which an amino group of the form —NH²or —NHR¹ (supra) is shielded by a protecting group. Suitable aminoprotecting groups include, but are not limited to, a carbamate, anamide, a sulfonamide, an imine, an imide, histidine, aN-2,5,-dimethylpyrrole, an N-1,1,4,4-tetramethyldisilylazacyclopentaneadduct, an N-1,1,3,3-tetramethyl-1,-disilisoindoline, anN-diphenylsilyldiethylene, an 1,3,5-dioxazine, a N-[2-(trimethylsilyl)ethoxy]methylamine, a N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,a N-4,4,4-trifluoro-3-oxo-1-butenylamine, a N-9-borabicyclononane and anitroamine. A protecting group may also be present that shields both theamino and the carboxylic group such as e.g. in the form of a2,2-dimethyl-4-alkyl-2-sila-5-oxo-1,3-oxazolidine. The alpha carbon atomof the amino acid typically further carries a hydrogen atom. The socalled “side chain” attached to the alpha carbon atom, which is in factthe continuing main chain of the carboxylic acid, is an aliphatic moietythat may be linear or branched. The term “side chain” refers to thepresence of the amino acid in a peptide (supra), where a backbone isformed by coupling a plurality of amino acids. An aliphatic moietybonded to the a carbon atom of an amino acid included in such a peptidethen defines a side chain relative to the backbone. As explained above,the same applies to an aliphatic moiety bonded to the amino group of theamino acid, which likewise defines a side chain relative to the backboneof a peptoid.

The term “aliphatic” means, unless otherwise stated, a straight orbranched hydrocarbon chain, which may be saturated or mono- orpoly-unsaturated and include heteroatoms. The term “heteroatom” as usedherein means an atom of any element other than carbon or hydrogen. Anunsaturated aliphatic group contains one or more double and/or triplebonds (alkenyl or alkynyl moieties). The branches of the hydrocarbonchain may include linear chains as well as non-aromatic cyclic elements.The hydrocarbon chain, which may, unless otherwise stated, be of anylength, and contain any number of branches. Typically, the hydrocarbon(main)chain includes 1 to 5, to 10, to 15 or to 20 carbon atoms.Examples of alkenyl radicals are straight-chain or branched hydrocarbonradicals which contain one or more double bonds. Alkenyl radicalsgenerally contain about two to about twenty carbon atoms and one ormore, for instance two, double bonds, such as about two to about tencarbon atoms, and one double bond. Alkynyl radicals normally containabout two to about twenty carbon atoms and one or more, for example two,triple bonds, preferably such as two to ten carbon atoms, and one triplebond. Examples of alkynyl radicals are straight-chain or branchedhydrocarbon radicals which contain one or more triple bonds. Examples ofalkyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, the n isomers of these radicals, isopropyl,isobutyl, isopentyl, sec-butyl, tert-butyl, neopentyl, 3, dimethylbutyl.Both the main chain as well as the branches may furthermore containheteroatoms as for instance N, 0, S, Se or Si or carbon atoms may bereplaced by these heteroatoms.

An aliphatic moiety may be substituted or unsubstituted with one or morefunctional groups. Substituents may be any functional group, as forexample, but not limited to, amino, amido, azido, carbonyl, carboxyl,keto, cyano, isocyano, dithiane, halogen, hydroxyl, nitro, organametal,organoboron, seleno, silyl, silano, sulfonyl, thio, thiocyano,trifluoromethyl sulfonyl, p-toluenesulfonyl, bromobenzenesulfonyl,nitrobenzenesulfonyl, and methanesulfonyl. As should be apparent fromthe above, the side chain of an amino acid in a peptide/peptoiddescribed herein may be of a length of 0 to about 5, to about 10, toabout 15 or to about 20 carbon atoms. It may be branched and includeunsaturated carbon-carbon bonds. In some embodiments one or more naturalamino acids are included in the peptide or peptoid. Such a natural aminoacid may be one of the 20 building blocks of naturally occurringproteins.

In a peptide or peptoid, including a peptide/peptoid disclosed hereinindividual amino acids are covalently coupled via amide bonds between acarboxylic group of a first and an amino group of a second amino acid. Apeptide and/or peptoid disclosed herein is non-repetitive, such that twoamino acids coupled to each other are always different from one another.

The term amphiphilic refers to a compound that is soluble in both polarand non-polar fluids. It also encompasses multiphase compounds. Theamphiphilic properties of the peptide and/or peptoid are due to thepresence of both polar and non-polar moieties within the same peptideand/or peptoid. In this regard the peptide and/or peptoid may be ofsurfactant nature. Accordingly, the polar properties of a peptide and/orpeptoid disclosed herein are based on a polar moiety. Two such moietyare a —COOH side group, in particular in the form of a charged COO—group and an amino group. A further such moiety is a C-terminal —COOHgroup if it is present in free, unprotected form. Generally, asurfactant molecule includes a polar, typically hydrophilic, head groupattached to a non-polar, typically hydrocarbon, moiety. Non-polarmoieties of a peptide or peptoid include a hydrocarbon chain that doesnot carry a functional group.

An amphiphilic linear sequence included in a peptide and/or peptoiddisclosed herein thus includes a polar moiety and a non-polar moiety.The polar moiety includes an aliphatic amino acid that carries a polargroup such as a hydroxyl group, a thiol group, a seleno group, an aminogroup, an amide group, an ether group, a thioether group or a selenoether group. Accordingly, the polar moiety may include an amino acidthat carries a functional polar group with a proton such as hydroxyl,thiol, selenol, amine or amide. The polar moiety may also include theC-terminus or the N-terminus of the peptide and/or peptoid. TheC-terminus or the N-terminus may in such a case be present in the formof the free carboxyl or amino group, respectively, i.e. free of aprotecting group.

Generally the polar moiety of a linear amphiphilic sequence of anamphiphilic peptide and/or peptoid disclosed herein is defined by asingle amino acid, by two consecutive amino acids or by threeconsecutive amino acids that is/are coupled to the non-polar moiety ofthe peptide/peptoid. Accordingly, in some embodiments the polar moietyof the peptide/peptoid consists of two amino acids that are covalentlycoupled via an amide bond, both amino acids carrying a polarpeptide/peptoid side chain. One of these two amino acids may be aterminal amino aid of the peptide/peptoid, defining its N- orC-terminus. In some embodiments the amphiphilic peptide/peptoid has asingle amino acid with a polar side chain with the residual portion ofthe peptide/peptoid defining the non-polar moiety. In some embodimentsthe amphiphilic peptide/peptoid has two amino acids with a polar sidechain while the residual portion of the peptide/peptoid defines thenon-polar moiety. As three illustrative examples of a respective polarside chain may serve 4-methyl-4-thio-pentyl,6-ethoxycarbonyl-4,5-dimethylhexyl and6-hydroxy-4-(1-hydroxyethyl)-hexyl groups. As used herein, the numberingof corresponding peptide/peptoid side chains is started with “1” at thecarbon atom that is covalently bonded to the a-carbon atom of the aminoacid or to the amino group of the amino acid, respectively. Amino acidsincluded in the polar moiety may be or include, but are not limited to,aspartic acid, asparagine, glutamic acid, 4-fluoro-glutamic acid,2-aminoadipic acid, γ-carboxy-glutamic acid, 4-tert-butyl aspartic acid,glutamine, 5-N-ethyl-glutamine (theanine), itrulline, thio-citrulline,cysteine, homocysteine, methionine, ethionine, selenomethionine,telluromethionine, threonine, allo-threonine, serine, homoserine,arginine, homoarginine, ornithine, lysine, 5-hydroxy-lysine andN(6)-carboxymethyllysine. Any such amino acid maybe present in the L- orD-form.

The amphiphilic linear sequence of the amphiphilic peptide/peptoiddisclosed herein can be defined as having n amino acids. Where a singleamino acid with a polar side chain is included in the amphiphilic linearsequence, the non-polar moiety may then be taken to have n−1 aminoacids. In this case the polar moiety consists of exactly one amino acid,such amino acid being selected from any amino acids of the foregoingparagraph. Where two consecutive amino acids with a polar side chain areincluded in the amphiphilic linear sequence of the peptide/peptoid, thenon-polar moiety may then be taken to have n−2 amino acids. In this casethe polar moiety consists of exactly two amino acids. Where threeconsecutive amino acids with a polar side chain are included in theamphiphilic linear sequence, the non-polar moiety may then be taken tohave n−3 amino acids. In this case the polar moiety consists of exactlythree amino acids. In embodiments where the polar moiety consists of twoamino acids, the polar moiety may have a sequence selected from Asn-Asn,Asp-Asp, Glu-Glu, Gln-Gln, Asn-Gln, Gln-Asn, Asp-Gin, Gin-Asp, Asn-Glu,Glu-Asn, Asp-Glu, Glu-Asp, Gln-Glu, Glu-Gln, Asp-Asn, Asn-Asp, Thr-Thr,Ser-Ser, Thr-Ser, Ser-Thr, Asp-Ser, Ser-Asp, Ser-Asn, Asn-Ser, Gln-Ser,Ser-Gln, Glu-Ser, Ser-Glu, Asp-Thr, Thr-Asp, Thr-Asn, Asn-Thr, Gin-Thr,Thr-Gln, Glu-Thr, hr-Glu. In embodiments where the polar moiety consistsof three amino acids, the polar moiety may have a sequence selected fromAsn-Asn-Asn, Asn-Asn-Asp, Asn-Asp-Asn, Asp-Asn-Asn, Asp-Asp-Asn,Asp-Asn-Asp, Asp-Asp-Asp, Asn-Asn-Glu, Asn-Asn-Gln, Asn-Glu-Asn,Asn-Gln-Asn, Glu-Glu-Glu, Gln-Gln-Gln, Asn-Gln-Gln, Asn-Glu-Gln,Asp-Asn-Glu, Gln-Asn-Asn, Gln-Asn-Asn, Glu-Asp-Gln, Asp-Gin-Asp,Asn-Glu-Asp, Glu-Asn-Gln, Asp-Glu-Gln, Asn-Glu-Gln, Glu-Asp-Asn, andGln-Asp-Asn, Thr-Thr-Thr, Ser-Ser-Ser, Asn-Thr-Thr, Asn-Ser-SerAsn-Ser-Thr, Asn-Thr-Ser Asp-Asn-Ser, Ser-Asn-Asn, Thr-Asn-Asn,Ser-Asp-Thr, to name a few.

The amphiphilic linear sequence of the peptide/peptoid has a net chargeat physiological pH. The term “physiological pH” is known to those inthe art to refer to the pH value of blood, which has typically a pHvalue of about 7.4. In embodiments where the amphiphilic linear sequenceis arranged at the C- or N-terminus of the peptide/peptoid, therespective terminus may provide the corresponding net charge. Inembodiments where the amphiphilic linear sequence is not arranged at theC- or N-terminus of the peptide/peptoid, the polar moiety of theamphiphilic linear sequence includes one or more amino acids that have aside chain with a functional group that is charged at physiological pH.Illustrative examples of a respective functional group include an amino,a nitro-, a guanidino, a esteryl, a sulfonyl or a carboxyl group. Insome embodiments the net charge of the amphiphilic linear sequence is,as a positive or as a negative charge, equal to or smaller than thenumber of amino acids included in the polar moiety thereof. In someembodiments the net charge of the amphiphilic linear sequence is one of−3, −2 or −1. In some embodiments the net charge of the amphiphiliclinear sequence is +1, +2 or +3.

The respective polar side chain of an amino acid of the polar moiety,coupled to the a-carbon atom of the amino acid (supra) and/or to theamino group thereof, may typically be defined by a main chain thatincludes 1 to about 20, including 1 to about 15, 1 to about 10 or 1 toabout 5 carbon atoms. For sake of clarity it is recited that the term“side chain” is used relative to the backbone of the peptide and/orpeptoid. This peptide and/or peptoid side chain may be branched and thusbe defined by a main chain and branches. Both the main chain andbranches, f present, of the peptide and/or peptoid side chain mayinclude one or more double or triple bonds (supra). Examples of sidechains include, but are not limited to, methyl, ethyl, propyl,isopropyl, propenyl, propinyl, butyl, butenyl, sec-butyl, tert-butyl,isobutyl, pentyl, neopentyl, sopentyl, pentenyl, hexyl, 3,3dimethylbutyl, heptyl, octyl, nonyl or decyl groups. The functionalpolar group is bonded to this the peptide and/or peptoid side chain. Insome embodiments the polar moiety of the amphiphilic linear sequenceincludes two identical amino acids. Where these amino acids arenaturally occurring amino acids, they may for example define one of thesequences Lys-Lys, Gln-Gln, Glu-Glu, Asp-Asp, Asn-Asn, Met-Met, Thr-Thr,Arg-Arg or Ser-Ser. The term “naturally occurring” in this contextrefers to the 20 amino acids into which the genetic code is directlybeing translated by any organism. Such two identical polar amino acidsmay for example be adjacent to the non-polar moiety. In some embodimentsthe amphiphilic linear sequence of the peptide/peptoid has a hydrophobictail of aliphatic amino acids and at least one polar, including acharged, amino acid head group. The non-polar moiety includes an aminoacid, generally at least two amino acids, with a hydrocarbon chain thatdoes not carry a functional group. The respective side chain, coupled tothe a-carbon atom of the amino acid (supra), may have a main chain thatincludes 0 to about 20 or 1 to about 20, including 0 to about 15, 1 toabout 15, 0 to about 10, 1 to about 10, 1 to about 5 or 0 to about 5carbon atoms.

The non-polar moiety may thus include an amino acid without side chain,i.e. glycine. The peptide and/or peptoid side chain may be branched(supra) and include one or more double or triple bonds (supra). Examplesof peptide and/or peptoid side chains include, but are not limited to,methyl, ethyl, propyl, isopropyl, propenyl, ropinyl, butyl, butenyl,sec-butyl, tert-butyl, isobutyl, pentyl, neopentyl, isopentyl, pentenyl,exyl, 3,3 dimethylbutyl, heptyl, octyl, nonyl or decyl groups. As a fewillustrative examples, the non-polar moiety may include an amino acid ofalanine, valine, leucine, isoleucine, norleucine, norvaline,2-(methylamino)-isobutyric acid, 2-amino-5-hexynoic acid. Such an aminoacid may be present in any desired configuration. Bonded to thenon-polar moiety may also be the C-terminus or the N-terminus of thepeptide/peptoid. Typically the C-terminus or the N-terminus is in such acase shielded by a protecting group (supra).

In some embodiments the non-polar moiety includes a sequence of aminoacids that is arranged in decreasing or increasing size. Hence, aportion of the amino acids of the non-polar moiety may be arranged in ageneral sequence of decreasing or increasing size. Relative to thedirection from N- to C-terminus or from C- to N-terminus this generalsequence can thus be taken to be of decreasing size. By the term“general sequence” of decreasing or increasing size is meant thatembodiments are included in which adjacent amino acids are of about thesame size as long as there is a general decrease or increase in size.Within a general sequence of decreasing size the size of adjacent aminoacids of the non-polar moiety is accordingly identical or smaller in thedirection of the general sequence of decreasing size. In someembodiments the general sequence of decreasing or increasing size is anon-repetitive sequence. As an illustrative example, where a respectiveportion of amino acids is a sequence of five amino acids, the firstamino acid may have a 3,-dimethyl-hexyl side chain. The second aminoacid may have a neopentyl side chain. The third amino acid may have apentyl side chain. The fourth amino acid may have a butyl side chain.The fifth amino acid may be glycine, i.e. have no side chain. Although aneopently and a pentyl side chain are of the same size, the generalsequence of such a non-polar peptide portion is decreasing in size. As afurther illustrative example of a general sequence of decreasing size ina non-polar moiety the respective non-polar portion may be a sequence ofthree amino acids. The first amino acid may have an n-nonyl side chain.The second amino acid may have a 3-ethyl-2-methyl-pentyl side chain. Thethird amino acid may have a tert-butyl side chain. As yet a furtherillustrative example of a general sequence of decreasing size in anon-polar moiety, the non-polar moiety may be a sequence of nine aminoacids. The first amino acid may have a 4-propyl-nonyl side chain. Thesecond amino acid may have an n-dodecyl side chain. The third amino acidmay have a 6,6-diethyl-3-octenyl side chain. An n-dodecyl side chain anda 6,6-diethyl-3-octenyl sidechain both have 12 carbon atoms and thusagain have a comparable size, Nevertheless, the 6,6-diethyl-3-octenylgroup includes an unsaturated carbon-carbon bond and is thus of slightlysmaller size than the dodecyl group. The fourth amino acid may have a2-methyl-nonyl sidechain. The fifth amino acid may have a 3-propyl-hexylside chain. The sixth amino acid may have an n-hexyl side chain. Theseventh amino acid may have a 2-butynyl side chain. The 8th amino acidmay have an isopropyl side chain. The ninth amino acid may have a methylsidechain.

Where a portion of the amino acids of the non-polar moiety arranged in ageneral sequence of decreasing (or increasing) size only containsnaturally occurring amino acids (whether in the D- or the L-form), itmay for example have a length of five amino acids, such as the sequenceleucine-isoleucine-valine-alanine-glycine orisoleucine-leucine-valine-alanine-glycine, A general sequence ofdecreasing size of only natural amino acids may also have a length offour amino acids. Illustrative examples include the sequencesisoleucine-leucine-valine-alanine, leucine-isoleucine-valine-alanine,isoleucine-valine-alanine-glycine, leucine-valine-alanine-glycine,eucine-isoleucine-alanine-glycine, leucine-isoleucine-valine-glycine,isoleucine-leucine-alanine-glycine or isoleucine-leucine-valine-glycine.A general sequence of decreasing size of only natural amino acids mayalso have a length of three amino acids. Illustrative examples includethe sequences isoleucine-valine-alanine, leucine-valine-alanine,isoleucine-valine-glycine, leucine-valine-glycine,leucine-alanine-glycine, isoleucine-alanine-glycine orisoleucine-leucine-alanine. A general sequence of decreasing size ofonly natural amino acids may also have a length of two amino acids.Illustrative examples include the sequences isoleucine-valine,leucine-valine, isoleucine-alanine, leucine-alanine, leucine-glycine,isoleucine-glycine, valine-alanine, valine-glycine or alanine-glycine.

In some embodiments the direction of decreasing size of the abovedefined general sequence of decreasing size is the direction toward thepolar moiety of the amphiphilic linear sequence. Accordingly, in suchembodiments the size of adjacent amino acids within this portion of thenon-polar moiety is accordingly identical or smaller in the direction ofthe polar moiety. Hence, as a general trend in such an embodiment, thecloser to the polar moiety of the amphiphilic linear sequence, thesmaller is the overall size of a peptide and/or peptoid side chainthroughout the respective general sequence of decreasing size. In theabove illustrative example of a general sequence of three amino acidswith a n-nonyl, a 3-ethyl-2-methyl-pentyl and a tert-butyl side chain,the next amino acid may be polar in that it carries a peptide/peptoidsidechain with a polar functional group. As an illustrative example,adjacent to the tert-butyl sidechain within the peptide/peptoid theremay be a 3-carboxy-n-butyl side chain.

In some embodiments the entire non-polar moiety of the amphiphiliclinear peptide and/or peptoid or the amphiphilic linear sequence,respectively, consists of the general sequence of decreasing (orincreasing) size. In such an embodiment the general sequence ofdecreasing (or increasing) size may have a length of n−m amino acids(cf. above). In some embodiments the general sequence of decreasing orincreasing size is flanked by further non-polar side chains of thepeptide/peptoid. In one embodiment the general sequence of decreasing(or increasing) size has a length of n−m−1 amino acids. In thisembodiment there is one further amino acid included in thepeptide/peptoid, providing a non-polar peptide/peptoid side chain. Thisamino acid may be positioned between the general sequence of decreasing(or increasing) size and the polar amino acid, the polar amino acid maybe positioned between this additional nonpolar amino acid and thegeneral sequence of decreasing (or increasing) size or the generalsequence of decreasing (or increasing) size may be positioned betweenthe polar amino acid and this additional non-polar amino acid. Typicallythe general sequence of decreasing (or increasing) size is positionedbetween the polar amino acid and this additional non-polar amino acid.The additional non-polar amino acid may for example define theN-terminus of the peptide/peptoid, which may be shielded by a protectinggroup such as an amide, e.g. a propionic acyl or an acetyl group.Together with the general sequence of decreasing (or increasing) size asdefined above it may define the non-polar portion of thepeptide/peptoid. The polar amino acid may define the C-terminus of thepeptide/peptoid. In this example the general sequence of decreasing (orincreasing) size is thus flanked by the polar amino acid on one side andby the additional non-polar amino acid on the other side. In oneembodiment where embodiment the general sequence of decreasing (orincreasing) size has a length of n−m−1 amino acids, the remainingnon-polar amino acid of the non-polar moiety of n−m amino acids is oneof alanine and glycine.

As explained above, the polar moiety of the amphiphilic linear sequencemay in some embodiments be defined by two or three consecutive aminoacids. The polar moiety includes m aliphatic amino acids. Each of the maliphatic amino acids is independently selected and carries anindependently selected polar group. The symbol m represents an integerselected from 1, 2 and 3. The at least essentially non-polar moiety(supra) accordingly has a number of n−m, i.e. n−1, n−2 or n−3 aminoacids. In some embodiments n is equal to or larger than m+2. In such anembodiment m may thus represent a number of n−2 or smaller.

In an embodiment where the entire non-polar moiety of the amphiphiliclinear peptide and/or peptoid consists of the general sequence ofdecreasing (or increasing) size (supra), this non polar moiety may thushave a length of n−2 or n−3 amino acids. In an embodiment where theamphiphilic linear peptide and/or peptoid has a further non-polar sidechain in addition to the non-polar moiety of decreasing (or increasing)size, this additional non-polar side chain maybe included in an aminoacid that is directly bonded to an amino acid of the general sequence ofdecreasing (or increasing) size. The non-polar moiety may thus bedefined by the non-polar moiety of decreasing (or increasing) size andthe respective further amino acid with a non-polar side chain. In onesuch an embodiment where m=1, the non-polar moiety may thus have alength of n−2 amino acids, of which the non-polar moiety of decreasing(or increasing) size has a length of n−3 amino acids. The generalsequence of decreasing (or increasing) size may be positioned betweenthe two polar amino acids and this additional non-polar amino acid, orthe additional non-polar amino acid may be positioned between thegeneral sequence of decreasing (or increasing) size and the two polaramino acids. Typically the general sequence of decreasing (orincreasing) size is positioned between the two polar amino acids andthis additional non-polar amino acid. As mentioned above, one of the twopolar amino acids may define the C-terminus of the peptide/peptoid. Inthis example the general sequence of decreasing (or increasing) size maythus be flanked by the two consecutive polar amino acids on one side andby the additional non-polar amino acid on the other side. Again, in someembodiments where m=1 the two consecutive polar amino acids may also bepositioned between the general sequence of decreasing (or increasing)size and the additional non-polar amino acid, in which case thenon-polar moiety has a first portion with a length of n−3 amino acidsand a further portion of one amino acid.

Electrostatic forces, hydrogen bonding and van der Waals forces betweenamphiphilic linear sequences as defined above, including amphiphiliclinear peptides and/or peptoids, result in these amphiphilic linearsequences to be coupled to each other. Without being bound by theory,hereby a cross-linking effect occurs that allows the formation of ahydrogel. In this regard the inventors have observed the formation offibers based on helical structures.

The fibers formed of amphiphilic linear sequences of amphiphilicpeptides and/or peptoids disclosed herein typically show high mechanicalstrength, which renders them particularly useful in tissue regenerationapplications, for instance the replacement of damaged tissue.Amphiphilic peptides and/or peptoids disclosed herein have been observedto generally assemble into a fiber structure that resembles collagenfibers. Collagen, a component of soft tissue in the animal and humanbody, is a fibrous protein that provides most of the tensile strength oftissue. The mechanical strength of fibers of amphiphilic peptides and/orpeptoids disclosed herein has been found to typically be much higherthan that of collagen (cf. e.g. Figures) of gelatine, the hydrolysedform of collagen. An amphiphilic peptide and/or peptoid disclosed hereinmay thus be included in a hydrogel that is used as permanent ortemporary prosthetic replacement for damaged or diseased tissue.

The amphiphilic linear sequence of the peptide/peptoid, which mayrepresent the entire amphiphilic peptide/peptoid (supra) has been foundto show remarkable stability at physiological conditions, even atelevated temperatures. It is in some embodiments stable in aqueoussolution at physiological conditions at ambient temperature for a periodof time in the range from 1 day to 1 month or more. It may in someembodiments be stable in aqueous solution at physiological conditions at90° C. for at least 1 hour, at least 2 hours, at least 3 hours, at least4 hours or at least 5 hours.

An amphiphilic linear sequence of an amphiphilic peptide and/or peptoiddisclosed herein, including an amphiphilic linear peptide and/orpeptoid, is capable of providing a self assembling a-helical fiber inaqueous solution under physiological conditions. The peptides/peptoids(typically 3-7-mers) in the L- or D-form can self assemble intosupramolecular helical fibers which are organized into mesh-likestructures mimicking biological substances such as collagen. It haspreviously been observed in X-ray crystallography that peptides of alength of 3 to 6 amino acids with repetitive alanine containingsequences and an acetylated C-terminus take a helical conformation(Hatakeyama, Y, et al, Angew. Chem. Int. Ed. (2009) 8695-8698). Usingpeptides with an amphiphilic sequence disclosed herein, Ac-LD₆ (L), theformation of aggregates has for example been observed already at 0.1mg/ml. As the concentration of peptide is increased to 1 mg/ml, thepeptide monomers were found to align to form fibrous structures. With aformation of fibers occurring under physiological conditions atconcentrations below 2 mM a peptide/peptoid disclosed herein is wellsuited as an injectable hydrogel material that can form a hydrogel underphysiological conditions. Also disclosed herein is an amphiphilic linearpeptide and/or peptoid as defined above for tissue engineering as wellas to a tissue engineering method that involves applying, includinginjecting a respective amphiphilic linear peptide and/or peptoid.

A hydrogel as disclosed herein is typically characterized by aremarkable rigidity and are generally biocompatible and non-toxic.Depending on the selected peptide/peptoid sequence these hydrogels canshow thermoresponsive or thixotropic character. Reliant on thepeptide/peptoid assembling conditions the fibers differ in thickness andlength. Generally rigid hydro gels are obtained that are well suited forcultivation of a variety of primary human cells, providingpeptide/peptoid scaffolds that can be useful in the repair andreplacement of various tissues. Disclosed is also a process of preparingthese hydrogels. The exemplary usage of these hydrogels in applicationssuch as cell culture, tissue engineering, plastic surgery, drugdelivery, oral applications, cosmetics, packaging and the like isdescribed, as well as for technical applications, as for example for usein electronic devices which might include solar or fuel cells.

As an amphiphilic linear sequence of the peptide/peptoid, a hydrogel asdisclosed herein shows high stability at physiological conditions, evenat elevated temperatures. In some embodiments such a hydrogel is stablein aqueous solution at ambient temperature for a period of at least 7days, at least 14 days, at least a month or more, such as at least 1 toabout 6 months.

In some embodiments a hydrogel disclosed herein is coupled to a moleculeor a particle, including a quantum dot, with characteristic spectral orfluorometric properties, such as a marker, including a fluorescent dye.A respective molecule may for instance allow monitoring the fate,position and/or the integrity of the hydrogel.

In some embodiments a hydrogel disclosed herein is coupled to a moleculewith binding affinity for a selected target molecule, such as amicroorganism, a virus particle, a peptide, a peptoid, a protein, anucleic acid, a peptide, an oligosaccharide, a polysaccharide, aninorganic molecule, a synthetic polymer, a small organic molecule or adrug.

The term “nucleic acid molecule” as used herein refers to any nucleicacid in any possible configuration, such as single stranded, doublestranded or a combination thereof. Nucleic acids include for instanceDNA molecules (e.g., DNA or genomic DNA), RNA molecules (e.g., RNA),analogues of the DNA or RNA generated using nucleotide analogues orusing nucleic acid chemistry, locked nucleic acid molecules (LNA), andprotein nucleic acids molecules (PNA). DNA or RNA may be of genomic orsynthetic origin and may be single or double stranded. In the presentmethod of an embodiment of the invention typically, but not necessarily,an RNA or a DNA molecule will be used. Such nucleic acid can be e.g.mRNA, cRNA, synthetic RNA, genomic DNA, eDNA synthetic DNA, a copolymerof DNA and RNA, oligonucleotides, etc. A respective nucleic acid mayfurthermore contain non-natural nucleotide analogues and/or be linked toan affinity tag or a label. In some embodiments the nucleic acidmolecule may be isolated, enriched, or purified. The nucleic acidmolecule may for instance be isolated from a natural source by eDNAcloning or by subtractive hybridization. The natural source may bemammalian, such as human, blood, semen, or tissue. The nucleic acid mayalso be synthesized, e.g. by the triester method or by using anautomated DNA synthesizer.

Many nucleotide analogues are known and can be used in nucleic acids andoligonucleotides used in the methods of exemplary embodiments of theinvention. A nucleotide analogue is a nucleotide containing amodification at for instance the base, sugar, or phosphate moieties.Modifications at the base moiety include natural and syntheticmodifications of A, C, G, and T/U, different purine or pyrimidine bases,such as uracil-5-yl, hypoxanthin-9-yl, and 2-aminoadenin-9-yl, as wellas non-purine or non-pyrimidine nucleotide bases. Other nucleotideanalogues serve as universal bases. Universal bases include3-nitropyrrole and 5-nitroindole. Universal bases are able to form abase pair with any other base. Base modifications often can be combinedwith for example a sugar modification, such as for instance2′-O-methoxyethyl, .g. to achieve unique properties such as increasedduplex stability.

A peptide may be of synthetic origin or isolated from a natural sourceby methods well known in the art. The natural source may be mammalian,such as human, blood, semen, or tissue. A peptide, including apolypeptide may for instance be synthesized using an automatedpolypeptide synthesizer. Illustrative examples of polypeptides are anantibody, a fragment thereof and a proteinaceous binding molecule withantibody-like functions. Examples of (recombinant)antibody fragments areFab fragments, Fv fragments, single-chain Fv fragments (scFv),diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409,437-441), decabodies (Stone, E., et al., Journal of ImmunologicalMethods (2007) 318, 88-94) and other domain antibodies (Holt, L. J., etal., Trends Biotechnol. (2003), 21, 11, 484-490). An example of aproteinaceous binding molecule with antibody-like functions is a muteinbased on a polypeptide of the lipocalin family (WO 03/029462, Beste etal., Proc. Natl. Acad. Sci. U.S.A. (1999)96, 1898-1903). Lipocalins,such as the bilin binding protein, the human neutrophilgelatinase-associated lipocalin, human Apolipoprotein D or glycodelin,posses natural ligand binding sites that can be modified so that theybind to selected small protein regions known as haptens. Examples ofother proteinaceous binding molecules are the so-called glubodies (seee.g. international patent application WO 96/23879), proteins based onthe ankyrin scaffold (Mosavi, L. K., et al., Protein Science (2004) 13,6, 1435-1448) or crystalline scaffold (e.g. international patentapplication WO 01/04144) the proteins described in Skerra, J. Mol.Recognit. (2000) 13, 167-187, AdNectins, tetranectins and avimers.Avimers contain so called A-domains that occur as strings of multipledomains in several cell surface receptors (Silverman, J., et al., NatureBiotechnology (2005) 23, 1556-1561). Adnectins, derived from a domain ofhuman fibronectin, contain three loops that can be engineered forimmunoglobulin-like binding to targets (Gill, D. S. & Damle, N. K.,Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins,derived from the respective human homotrimeric protein, likewise containloop regions in a C-type lectin domain that can be engineered fordesired binding (ibid.). Where desired, a modifying agent may be usedthat further increases the affinity of the respective moiety for any ora certain form, class etc. of target matter.

An example of a nucleic acid molecule with antibody-like functions is anaptamer. An aptamer folds into a defined three-dimensional motif andshows high affinity for a given target structure. Using standardtechniques of the art such as solid-phase synthesis an aptamer withaffinity to a certain target can accordingly be formed and immobilizedon a hollow particle of an embodiment of the invention.

As a further illustrative example, a linking moiety such as an affinitytag may be used to immobilise the respective molecule. Such a linkingmoiety may be a molecule, e.g. a hydrocarbon-based (including polymeric)molecule that includes nitrogen-, phosphorus-, sulphur-, arben-,halogen- or pseudohalogen groups, or a portion thereof. As anillustrative example, the peptide/peptoid included in the hydrogel mayinclude functional groups, for instance on aside chain of thepeptide/peptoid, that allow for the covalent attachment of abiomolecule, for example a molecule such as a protein, a nucleic acidmolecule, a polysaccharide or any combination thereof. A respectivefunctional group may be provided in shielded form, protected by aprotecting group that can be released under desired conditions. Examplesof a respective functional group include, but are not limited to, anamino group, an aldehyde group, a thiol group, a carboxy group, anester, an anhydride, a sulphonate, a sulphonate ester, an imidoester, asilyl halide, an epoxide, an aziridine, a phosphoramidite and adiazoalkane. Examples of an affinity tag include, but are not limitedto, biotin, dinitrophenol or digoxigenin, ligohistidine, polyhistidine,an immunoglobulin domain, maltose-binding protein,glutathione-S-transferase (GST), calmodulin binding peptide (CBP),FLAG′-peptide, the T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly),maltose binding protein (MBP), the HSV epitope of the sequenceGln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virusglycoprotein D, the hemagglutinin (HA) epitope of the sequenceTyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, the “myc” epitope of thetranscription factor c-myc of the sequenceGlu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, or an oligonucleotide tag. Suchan oligonucleotide tag may for instance be used to hybridise to animmobilised oligonucleotide with a complementary sequence. A furtherexample of a linking moiety is an antibody, a fragment thereof or aproteinaceous binding molecule with antibody-like functions (see alsoabove). A further example of linking moiety is a cucurbituril or amoiety capable of forming a complex with a cucurbituril. A cucurbiturilis a macrocyclic compound that includes glycoluril units, typicallyself-assembled from an acid catalyzed condensation reaction ofglycoluril and formaldehyde. A cucurbit[n]uril, (CB[n]), that includes nglycoluril units, typically has two portals with polar ureido carbonylgroups. Via these ureido carbonyl groups cucurbiturils can bind ions andmolecules of interest. As an illustrative example cucurbit[7]uril(CB[7]) can form a strong complex with ferrocenemethylammonium oradamantylammonium ions. Either the cucurbit[7]uril or e.g.ferrocenemethylammonium may be attached to a biomolecule, while theremaining binding partner (e.g. ferrocenemethylammonium orcucurbit[7]uril respectively) can be bound to a selected surface.Contacting the biomolecule with the surface will then lead to animmobilisation of the biomolecule. Functionalised CB[7] units bound to agold surface via alkanethiolates have for instance been shown to causean immobilisation of a protein carrying a ferrocenemethylammonium unit(Hwang, I., et al., J. Am. Chem. Soc. (2007) 129, 4170-4171).

Further examples of a linking moiety include, but are not limited to anoligosaccharide, anoligopeptide, biotin, dinitrophenol, digoxigenin anda metal chelator (cf. also below). As an illustrative example, arespective metal chelator, such as ethylenediamine,ethylenediaminetetraaceticacid (EDTA), ethylene glycol tetraacetic acid(EGTA), diethylenetriaminepentaaceticacid (DTPA),N,N-bis(carboxymethyl)glycine (also called nitrilotriacetic acid, NTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),2,3-dimercapto-1-propanol (dimercaprol), porphine or heme may be used incases where the target molecule is a metal ion. As an example, EDTAforms a complex with most monovalent, divalent, trivalent andtetravalent metal ions, such as e.g. silver (Ag⁺), calcium (Ca²⁺),manganese (Mn²⁺), copper (Cu²⁺), iron (Fe²⁺), cobalt (Co³⁺) andzirconium (Zr⁴⁺), while BAPTA is specific for Ca²⁺. In some embodimentsa respective metal chelator in a complex with a respective metal ion ormetal ions defines the linking moiety. Such a complex is for example areceptor molecule for a peptide of a defined sequence, which may also beincluded in a protein. As an illustrative example, a standard methodused in the art is the formation of a complex between an oligo histidinetag and copper (Cu²⁺), nickel (Ni²⁺), cobalt (Co²⁺), or zink (Zn²⁺)ions, which are presented by means of the chelator nitrilotriacetic acid(NTA).

Avidin or streptavidin may for instance be employed to immobilise abiotinylated nucleic acid, or a biotin containing monolayer of gold maybe employed (Shumaker-Parry, J. S., et al., Anal. Chem. (2004) 76, 918).As yet another illustrative example, the biomolecule may be locallydeposited, e.g. by scanning electrochemical microscopy, for instance viapyrroleoligonucleotide patterns (e.g. Fortin, E., et al.,Electroanalysis (2005) 17, 495). In other embodiments, in particularwhere the biomolecule is a nucleic acid, the biomolecule may be directlysynthesised on the surface of the immobilisation unit, for example usingphotoactivation and deactivation.

As an illustrative example, the synthesis of nucleic acids oroligonucleotides on selected surface areas (so called “solid phase”synthesis) may be carried out using electrochemical reactions usingelectrodes. An electrochemical deblocking step as described by Egeland &Southern (Nucleic Acids Research (2005) 33, 14, e125) may for instancebe employed for this purpose. A suitable electrochemical synthesis hasalso been disclosed in US patent application US 2006/0275927. In someembodiments light-directed synthesis of a biomolecule, in particular ofa nucleic acid molecule, including UV-linking or light dependent5′-deprotection, may be carried out.

The molecule that has a binding affinity for a selected target moleculemay be immobilised on the nanocrystals by any means. As an illustrativeexample, an oligo- or polypeptide, including a respective moiety, may becovalently linked to the surface of nanocrystals via a thio-ether bond,or example by using ω functionalized thiols. Any suitable molecule thatis capable of linking a nanocrystal of an embodiment of the invention toa molecule having a selected binding affinity may be used to immobilisethe same on a nanocrystal. For instance a (bifunctionalμinking agentsuch as ethyl-3-dimethylaminocarbodiimide, N-(3-aminopropyl)3-mercaptobenzamide, 3-aminopropyl-trimethoxysilane,3-mercaptopropyl-trimethoxysilane, 3-(trimethoxysilyl) propyl-maleimide,or 3-(trimethoxysilyl) propyl-hydrazide may be used. Prior to reactionwith the linking agent, the surface of the nanocrystals can be modified,for example by treatment with glacial mercaptoacetic acid, in order togenerate free mercaptoacetic groups which can then employed forcovalently coupling with an analyte binding partner via linking agents.

Embodiments of the present invention also include a hydrogel, which canbe taken to be a water-swollen water-insoluble polymeric material. Thehydrogel includes, including contains and consists of, a peptide and/orpeptoid as defined above. Since a hydrogel maintains a three dimensionalstructure, a hydrogel of an embodiment of the invention may be used fora variety of applications. Since the hydrogel has a high water contentand includes amino acids, it is typically of excellent biocompatibility.

A hydrogel according to an embodiment of the invention is formed byself-assembly. The inventors have observed that the peptides/peptoidsassemble into fibers that form mesh-like structures. Without being boundby theory hydrophobic interaction between non-polar portions ofpeptides/peptoids as disclosed herein are contemplated to assist suchself-assembly process.

The method of forming the hydrogel includes dissolving thepeptide/peptoid in aqueous solution. Agitation, including mixing such asstirring, and/or sonication may be employed to facilitate dissolving thepeptide/peptoid. In some embodiments the aqueous solution with thepeptide/peptoid therein is exposed to a temperature below ambienttemperature, such as a temperature selected from about 2° C. to about15° C. In some embodiments the aqueous solution with the peptide/peptoidtherein is exposed to an elevated temperature, i.e. a temperature aboveambient temperature. Typically the aqueous solution is allowed to attainthe temperature to which it is exposed. The aqueous solution may forexample be exposed to a temperature from about 25° C. to about 85° C. orhigher, such as from about 25° C. to about 75° C., from about 25° C. toabout 70° C., from about 30° C. to about 70° C., from about 35° C. toabout 70° C., from about 25° C. to about 60° C., from about 30° C. toabout 60° C., from about 25° C. to about 50° C., from about 30° C. toabout 50° C. or from about 40° C. to about 65° C., such as e.g. atemperature of about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C. or about 65° C. The aqueous solution with thepeptide/peptoid therein may be maintained at this temperature for aperiod of about 5 min to about 10 hours or more, such as about 10 min toabout 6 hours, about 10 min to about 4 hours, about 10 min to about 2.5hours, about 5 min to about 2.5 hours, about 10 min to about 1.5 hoursor about 10 min to about 1 hour, such as about 15 min, about 20 min,about 25 min, about 30 min, about 35 min or about 40 min.

A hydrogel according to an embodiment of the invention may be includedin a fuel cell, here it may for example provide a substrate between theanode and the cathode. A liquid electrolyte may be encompassed by thehydrogel. Likewise, a hydrogel according to an embodiment of theinvention may provide a substrate between two electrodes in anelectrophoresis apparatus. The hydrogel may also be conducting. Thehydrogel may also serve in enhancing the efficiency of charge-separatedstates and/or slowing down charge recombination. The hydrogel may thusbe applied in any form photovoltaics, including a solar cell.

In some embodiments a hydrogel disclosed herein is a biocompatible,including a pharmaceutically acceptable hydrogel. The term“biocompatible” (which also can be referred to as “tissue compatible”),as used herein, is a hydrogel that produces little if any adversebiological response when used in vivo. The term thus generally refers tothe inability of a hydrogel to promote a measurably adverse biologicalresponse in a cell, including in the body of an animal, including ahuman. A biocompatible hydrogel can have one or more of the followingproperties: non-toxic, non-mutagenic, non-allergenic, non-carcinogenic,and/or non-irritating. A biocompatible hydrogel, in the least, can beinnocuous and tolerated by the respective cell and/or body. Abiocompatible hydrogel, by itself, may also improve one or morefunctions in the body.

Depending on the amino acids that are included in the peptide/peptoidthat is included in a hydrogel, a respective hydrogel may bebiodegradable. A biodegradable hydrogel gradually disintegrates or isabsorbed in vivo over a period of time, e.g., within months or years.Disintegration may for instance occur via hydrolysis, may be catalysedby an enzyme and may be assisted by conditions to which the hydrogel isexposed in a human or animal body, including a tissue, a blood vessel ora cell thereof. Where a peptide is made up entirely of natural aminoacids, a respective peptide can usually be degraded by enzymes of thehuman/animal body.

A hydrogel according to an embodiment of the invention may also serve asa depot for a pharmaceutically active compound such as a drug. Ahydrogel according to an embodiment of the invention may be designed tomimic the natural extracellular matrix of an organism such as the humanor animal body. A fiber formed from the peptide/peptoid of an embodimentof the invention, including a respective hydrogel, may serve as abiological scaffold. A hydrogel of an embodiment of the invention may beincluded in an implant, in a contact lens or may be used in tissueengineering. In one embodiment, the peptides consist typically of 3-7amino acids and are able to self-assemble into complex fibrous scaffoldswhich are seen as hydrogels, hen dissolved in water or aqueous solution.These hydrogels can retain water up to 99.9% and possess sufficientlyhigh mechanical strength. Thus, these hydrogels can act as artificialsubstitutes for a variety of natural tissues without the risk ofimmunogenicity. The hydrogels in accordance with the present inventionmay be used for cultivating suitable primary cells and thus establish aninjectable cell-matrix compound in order to implant or reimplant thenewly formed cell-matrix in vivo. Therefore, the hydrogels in accordancewith the present invention are particularly useful for tissueregeneration or tissue engineering applications. As used herein, areference to an “implant” or “implantation” refers to uses andapplications of/for surgical or arthroscopic implantation of a hydrogelcontaining device into a human or animal, e.g. mammalian, body or limb.Arthroscopic techniques are taken herein as a subset of surgicaltechniques, and any reference to surgery, surgical, etc., includesarthroscopic techniques, methods and devices. A surgical implant thatincludes a hydrogel according to an embodiment of the invention mayinclude a peptide and/or peptoid scaffold. This the peptide and/orpeptoid scaffold may be defined by the respective hydrogel. A hydrogelof an embodiment of the invention may also be included in a wound coversuch as gauze or a sheet, serving in maintaining the wound in a moiststate to promote healing.

Depending on the amino acid sequence used in the peptide/peptoid thehydrogel may be temperature-sensitive. It may for instance have a lowercritical solution temperature or a temperature range corresponding tosuch lower critical solution temperature, beyond which the gel collapsesas hydrogen bonds by water molecules are released as water molecules arereleased from the gel.

The disclosed subject matter also provides improved chiral amphiphilicnatural-based peptides and/or peptoids that assemble to peptide/peptoidhydrogels with very favorable material properties. The advantage ofthese peptide/peptoid hydrogels is that they are accepted by a varietyof different primary human cells, thus providing peptide scaffolds thatcan be useful in the repair and replacement of various tissues.Depending on the chirality of the peptide monomer the character of thehydro gels can be designed to be more stable and less prone todegradation though still biocompatible.

A hydrogel and/or a peptide/peptoid described herein can be administeredto an organism, including a human patient per se, or in pharmaceuticalcompositions where it may include or be mixed with pharmaceuticallyactive ingredients or suitable carriers or excipient(s). Techniques forformulation and administration of respective hydro gels orpeptides/peptoids resemble or are identical to those of low molecularweight compounds well established in the art. Exemplary routes include,but are not limited to, oral, transdermal, and parenteral delivery. Ahydrogel or a peptide/peptoid may be used to fill a capsule or tube, ormay be provided in compressed form as a pellet. The peptide/peptoid orthe hydrogel may also be used in injectable or sprayable form, forinstance as a suspension of a respective peptide/peptoid.

A hydrogel of an embodiment of the invention may for instance be appliedonto the skin or onto a wound. Further suitable routes of administrationmay, for example, include depot, oral, rectal, transmucosal, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intravenous, intramedullary injections, as well asintrathecal, direct intraventricular, intraperitoneal, intranasal, orintraocular injections. It is noted in this regard that foradministering microparticles a surgical procedure is not required. Wherethe microparticles include a biodegradable polymer there is no need fordevice removal after release of the anti-cancer agent. Nevertheless themicroparticles may be included in or on a scaffold, a coating, patch,composite material, a gel or a plaster.

In some embodiments one may administer a hydrogel and/or apeptide/peptoid in a local rather than systemic manner, for example, viainjection.

Pharmaceutical compositions that include a hydrogel and/or apeptide/peptoid of an embodiment of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with an embodiment ofthe present invention thus may be formulated in conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries that facilitate processing of the hydrogeland/or peptide/peptoid into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For injection, the peptide/peptoid of an embodiment of the invention maybe formulated in aqueous solutions, for instance in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the hydrogel and/or peptide/peptoid can beformulated readily by combining them with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the hydrogel and/orpeptide/peptoid, as well as a pharmaceutically active compound, o beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a patient tobe treated. Pharmaceutical preparations for oral use can be obtained byadding a solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatine, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatine, as well as soft, sealed capsules made ofgelatine and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the peptides/peptoids may be suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for such administration.For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The hydrogel and/or peptide/peptoid may be formulated for parenteraladministration by injection, .g., y intramuscular injections or bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The respective compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulator agents such as suspending,stabilizing and/or dispersing agents. The hydrogel and/orpeptide/peptoid may be formulated for other drug delivery systems likeimplants, or trandermal patches or stents.

Examples

Experiments have been performed to illustrate the technical aspects ofexemplary embodiments of the present invention. The following examplesare described in the Experimental Methods and Results. The skilledartisan will readily recognize that the examples are intended to beillustrative and are not intended to limit the scope of the presentinvention.

Experimental Methods and Results

Peptides

The peptide sequences were designed to represent an amphiphilic peptidestructure containing a hydrophilic head group and a hydrophobic tail.The rationale for the peptides design was to create a peptide monomer ofdecreasing size resembling a cone shaped structure. The hydrophobic taildiffers by using different aliphatic amino acids. It is consisting ofthe following aliphatic amino acids such as glycine, alanine, valine,leucine and isoleucine and the hydrophilic head group is consisting ofone or two polar or charged amino acids. The sequence order of thehydrophobic tail differed by using different aliphatic amino acids. Thepeptides were commercially synthesized from GL Biochem, Shanghai, China.In order to verify the reproducibility of the peptide hydro gel-formingbehavior peptides were also synthesized from other companies (BiomatikCorp., Anaspec. Inc, USA). The peptides have a purity of equal or higherthan 95% verified by High-performance liquid chromatography (HPLC) andmass spectrometry. The peptide stock solutions were dissolved in waterat 5 to 10 mg/ml. Most of the peptides are acetylated at the N-terminus.

Peptide-Based Hydrogel Preparation

All peptides (GL Biochem, Shanghai, China, ≥98% purity) were freshlyprepared in order to avoid premature peptide aggregation. The peptideswere dissolved in water and left at room temperature to form hydrogels.Depending on the peptide concentration, the self-assembly processoccurred immediately, within hours or even within days (experimentaltime frame for gelation). For higher peptide concentrations peptideswere dissolved in milliQ water by vortexing. If a forced and acceleratedhydrogel preparation was needed, the peptide solution was subjected tosonication in a water bath (Barnstead Labline 9319 UltrasonicLC60H). Nosignificant structural differences were observed between hydrogelsproduced via self-assembly and those whose assembly was facilitated bysonication. Few peptides formed hydrogels more easily at elevatedtemperatures, i.e. at 50° C.

To study the effect of concentration variation, both AcLD₆ (L) and AciD₃(L) hydrogels were prepared with varying concentration as specifiedabove. To study the effect of monovalent and divalent cations, AcLD₆ (L)hydrogels were prepared by dissolving peptide in 10, 50, 100 and 150 mMNaCl and CaCl₂ solutions. FESEM and rheology studies were furtherperformed to characterize the morphology and strength of these hydrogels.

Preparation of gelatin and collagen gels: Gelatin (Type A, G 1890; SigmaAldrich) hydrogels was prepared by first dissolving gelatin in milli Qwater by heating followed by cooling till the gelation was observed.Collagen (Type I from bovine, Advanced Biomatrix, USA) was diluted withPBS buffer to a concentration of 1.5 mg/ml and titrated to pH 7.4 using0.1M NaOH. Gelation was achieved by incubating the solution at 37° C.for 1 hour.

Circular Dichroism (ω) Spectroscopy

Secondary peptide structures were analyzed by measuring ellipticityspectra using the Aviv Circular Dichroism Spectrometer, model 410. ωsamples were prepared by diluting stock peptides solutions (5-10 mg/ml)in water. The diluted peptide solutions were filled in to a cuvette with1 mm path length and spectra were acquired. As a blank reference waterwas used and the reference was subtracted from the raw data before molarellipticity was calculated. The calculation was based on the formula:[θ]λ=θ_(obs)× 1/(10 Lcn), where [θ]λ the molar ellipticity at λ, in degcm² d/mol, is the observed ellipticity at λ in mdeg, L is the pathlength in cm, c is the concentration of the peptide in M, and n is thenumber of amino acids in the peptide. Secondary structure analysis wasdone using CDNN software.

Environmental Scanning Electron Microscopy (ESEM)

Samples were placed onto a sample holder of FEI Quanta 200 EnvironmentalScanning Electron Microscopy. The surface of interest was then examinedusing accelerating voltage of 10 kV at a temperature of 4° C.

Field Emission Scanning Electron Microscopy (FESEM)

Samples were frozen at −20° C. and subsequently to −80° C. Frozensamples were further freeze dried. Freeze dried samples were fixed ontoa sample holder using conductive tape and sputtered with platinum fromboth the top and the sides in a JEOL JFC-1600 High Resolution SputterCoater. The coating current used was 30 mA and the process lasted for 60sec. The surface of interest was then examined with a JEOL JSM-7400FField Emission Scanning Electron Microscopy system using an acceleratingvoltage of 5-10 kV.

Rheological Measurements

To determine the viscoelastic properties of the peptide-based hydrogels, hydro gels were subjected to dynamic time, strain and frequencysweep experiments using the ARES-G2 rheometer (TA Instruments,Piscataway, N.J.) with the 25.0 mm diameter titanium parallel plategeometry and a 0.8 mm gap distance. Oscillatory frequency study wasperformed to compare the strength of peptide based hydrogel with varyingconcentration of peptides, or for peptide in presence of monovalent ordivalent ions. Oscillatory frequency sweep studies were performed at0.1-100 rad/s frequency and 0.1% strain at 25° C. and 50° C.

Ac-LD₆ [L]:

Peptide Sequence:

Ac-LIVAGD-COOH

Molecular weight: 629.56

(1) Temperature sweep study for Ac-LD₆ (L):

-   -   (a) The peptide mixture was then placed on rheometer lower        plate. Following parameters were optimized:    -   Gap between two plates: 1 mm    -   Strain: 10%    -   Frequency: 6.28 rad/sec    -   Temperature scan: 4° C. to 60° C.    -   Sample volume: 500 μl        (2) Frequency sweep study for Ac-LD₆(L):    -   Optimized parameter required to perform frequency sweep study    -   Gap between two plates: 0.8 mm    -   Strain: 0.1%    -   Temperature: 25 and 50° C.    -   Sample volume: 1 ml    -   Frequency scan: 0.1 rad/sec to 100 rad/sec    -   Concentration of Ac-LD-6 (L) in hydrogel: 10 mg/ml        (3) Effect of concentration variation of Ac-LD₆ (L) on gel        strength:    -   Optimized parameters that are required to perform frequency        sweep studies for measuring gel strength are as follows:    -   Gap between two plates: 0.8 mm    -   Strain: 0.1%    -   Temperature: 25 and 50° C.    -   Sample volume: 1 ml    -   Frequency scan: 0.1 rad/sec to 100 rad/sec    -   Concentrations of Ac-LD₆ (L) in hydrogels: 5 mg/ml, 10 mg/ml, 15        mg/ml and, 0 mg/ml and 30 mg/ml in water.        (4) Effect of sodium chloride (NaCl) on the gel strength of        Ac-LD₆ (L):

Effect of sodium chloride on Ac-LD₆ (L) based hydrogels, were studied byperforming a frequency sweep study on hydro gels prepared by dispersing10 mg of Ac-LD-6 (L) in varying concentration of NaCl solution forexample 10 mM, 50 mM, 100 mM and 150 mM of NaCl solution using optimizedprocedure to form hydro gels. Optimized parameter required to performfrequency sweep study to measure gel strength in presence of NaCl are asfollows:

-   -   Gap between two plates: 0.5 mm and 0.8 mm    -   Strain: 10% and 0.1% respectively    -   Temperature: 25° C. and 50° C.    -   Sample volume: 1 ml    -   Frequency scan: 0.1 rad/sec to 100 rad/sec    -   Concentrations of NaCl solutions used to prepare 10 mg/ml of        Ac-LD-6 (L) Hydrogels: 10 mM, 50 mM, 100 mM, 150 mM NaCl        solution.        Cell Growth Experiments

In order to find out whether the peptide hydrogels can serve as ascaffold for tissue engineering, its biocompatibility was investigated.Different primary human cells were seeded on top of the hydrogel afterits gelation in tissue culture medium (DMEM without serum) in 6-well,4-well or 96-well culture plates, see the culture conditions below.During the next 2-4 days no change of medium was necessary, buteventually fresh media was added to the wells. The cells were analyzedfor viability.

Primary human renal proximal tubule cells (HPTCs) and primary humanumbilical vein endothelial cells (HUVECs) were obtained from ScienCellResearch Laboratories (Carlsbad, Calif., SA). HPTCs were cultivated inbasal epithelial cell medium supplemented with 2% fetal bovine serum(FBS) and 1% epithelial cell growth supplement (all components obtainedfrom ScienCell Research Laboratories). The culture medium for HUVECs wasendothelial cell medium containing 5% FBS and 1% endothelial cell growthsupplement (ScienCell Research Laboratories). All cell culture mediaused were supplemented with 1% penicillin/streptomycin solution(ScienCell Research Laboratories), and all cells were cultivated at 37°C. in a 5% CO₂ atmosphere. The seeding density of the cells was about5×10⁴ cells/cm². However since HUVECs are bigger than HPTCs the cellnumber would be slightly lower than one for HPTC cells (˜4.5×10⁴cells/cm²). Both cell types had a confluency of about 80% in the wellsafter seeding.

Crosslinked Hydrogels

Material & Methods

Peptides

All peptides were synthesised at American Peptide Company (CA, USA)using SPPS and purified to >95% (HPLC). Amino acid content (AA %)analysis was performed and the net weight (gross weight×AA %) was usedfor calculations.

Kinetics of Disulfide Formation

For air oxidation, LK₆C was dissolved in MilliQ water by vigorousvortexing for five minutes and dispensed into 20 μL aliquots. Atappropriate time points, 180 μL of DTNB (Sigma, Singapore) workingsolution (4 mg/mL in 0.1M phosphate buffer pH 7.0) was mixed with thepeptide solution for 15 minutes. Absorbance at 412 nm was then measured(InfiniteM200, Tecan, Switzerland). Using a calibration curve generatedwith L-cysteine (Sigma) as standard (R²=0.999), thebackground-subtracted value was normalised to the reading at 0 hour togive the % of thiol remaining. For H₂O₂-assissted oxidation, LK₆C wasdissolved in water (HPLC grade, J. T. Baker, NJ, USA) containing 0.06%H₂O₂ (Merck, Sinagpore) and dispensed into aliquots. At appropriate timepoints, the aliquots were analysed using an Aquity® UPLC (Waters, SA)fitted with a single-quadrupole MS. Using a calibration curve generatedwith pure LK₆C as standard (R²=0.999), the area under the peakcorresponding to LK₆C monomer was normalised to that at 0 hour to givethe % of thiol remaining.

Gel Casting

Peptide was dissolved in 200 μL of water+/−H₂O₂ and filled intocustom-made hollow ring casts (diameter ˜1 cm). The ring ends weresealed with parafilm to minimise evaporation and the cast was kept at25° C. for 22 hours before further manipulations.

Rheology: The rheological properties of casted gels were measured withan ARES-G2 (TA Instruments, USA) using the oscillation method.Frequency-sweep studies were performed with ω=0-100 rad/s at strain,γ=0.1%. The gel stiffness was represented by plotting G′ against ω.Amplitude-sweep studies were performed beforehand at 1 Hz with y=0-100%.The LVE limit of the gel was defined as the value of γ when G′ firstdropped below 90% of the average initial value.

Rheology

The rheological properties of casted gels were measured with an ARES-G2(TA Instruments, SA) using the oscillation method. Frequency-sweepstudies were performed with ω=0-100 rad/s at strain, γ=0.1%. The gelstiffness was represented by plotting G′ against ω. Amplitude sweepstudies were performed beforehand at 1 Hz with γ=0-100%. The LVE limitof the gel was defined as the value of γ when G′ first dropped below 90%of the average initial value.

FESEM

Freeze-dried gels were deposited onto carbon tapes, sputtered withplatinum and observed under a JSM-7400F electron microscope (Jeol,Tokyo, Japan).

3D Cell Culture

LK₆C+/−CRGD gels were casted directly into 8-chamber Lab-Tek® wells(Nunc, N.Y., USA) and purified as reported in the SI. 0.5 mL of cellsuspension in regular completed DMEM (Invitrogen, Singapore) was eitherseeded onto the gel or directly into the well (2D control) and incubatedfor four days. 0.5 μL of calcein-AM (Invitrogen) was then added to themedia to identify live cells and images were captured using a confocalmicroscope (LSM5 DUO, Carl Zeiss, Germany). Beforehand, calcein-AM wasverified to stain only live cells as ethanol- and H₂O₂-treated cellsexcluded the dye. All images, except for the 2D controls, were presentedas mergers of several z-stacks. To further verify the 3D distribution ofcells, the gel was embedded vertically in Jung Tissue Freezing Medium(Leica Instruments GmbH, Germany) and 20 μm cross-sectional slices wereobtained with a cryostat (CM3050 S, Leica). Sections were mounted withcoverslips and imaged as above.

Quantification of Cell-Spread Area

Confocal images after calcein staining were processed using ImageJ (NIH,USA) to obtain the average cell-spread area under various cultureconditions. For cells in 3D gel culture, images used for quantificationwere mergers of several z-stacks to capture the maximum cell-spreadarea. In all cases, at least three independent experiments with at leasttwo locations selected from each culture were used for averaging.

MTT Viability Assay

After four days of 3D gel culture, cells were separated from thesubstrate by trypsination and centrifugation and re-seeded into 48-wellplates (Nunc) overnight for attachment. MTT (Sigma) dissolved in PBS wasthen added to the media for four hours, before the formazan crystalswere dissolved using DMSO (Sigma). Absorbance was read at 560 nm,subtracted with the 680 nm reference and normalised to the appropriatecontrol to give an indication of relative cell viability.

Statistical Analysis

ANOVA testing (OriginLab Corporation, MA, USA) was performed on samplemeans with p<0.05 being denoted by * and accepted to be statisticallysignificant.

Results

LK₆C Peptides can be Casted into 3-Dimensional Shapes

LK₆C peptides can be easily casted into 3-dimensional gels using theappropriate moulds (see FIG. 18). In this experiment, a mould in theshape of a ring was used and LK₆C gels were casted using water orcompleted growth medium. Dextran particles, employed here as a modelcargo, could also be encapsulated within the gel matrix.

H₂O₂-Assisted Formation of Disulfide Crosslinks

The kinetics of disulfide formation under air oxidation is inefficient(FIG. 19). The addition of H₂O₂ speeds up disulfide formation (FIG. 20).LC-MS confirms the formation of disulfide-crosslinked dimers in thepresence of H₂O₂ (FIG. 21). The data further suggests that the additionof HRP has no significant effect on the kinetics of disulfide formation(FIG. 19). The rate of disulfide formation can also be accelerated byincreasing the amount of H₂O₂ used (FIG. 22).

Oxidation Strategy

Two different methods were devised for the H₂O₂-assisstedcross-linking: 1) The cast-and-soak method, where the gel is first beingcasted overnight before being soaked in an oxidative solution. 2) The insitu oxidation method where the aqueous solution used to dissolve thepeptide powder already contains the oxidative agent (FIG. 23).

Oxidation Increases the Long-Term Stability of LK₆C in Water

LK₆ or LK₆C gels were first casted and then soaked in water for variousdurations. After 24 hours of water soak, the non-crosslinkable LK6 gelswere completely degraded while the non-oxidised LK₆C gels were severelydegraded (FIG. 24). In stark contrast, LK₆C gels oxidised for 2 hoursusing the cast-and-soak method and LK₆C gels oxidised in situ remainedintact after 96 hours of water soak. These observations are alsosupported by rheological measurements (FIG. 28). Therefore, the strategyof disulfide cross-linking has increased the resistance of the gel todegradation and has made it possible to use the gel at a lowerconcentration compared to previous formulations. This, in turn, willtranslate into significant cost savings.

Gel Oxidised Using the Cast-and-Soak Method is Uniformly Oxidised

LK₆C gels were casted overnight and then soaked in H₂O₂ solution for 2hours (see scheme in FIG. 25). Then the surfaces(circumference+top+bottom layers) were separated from the core and itwas shown that the amount of H₂O₂ and disulfide/thiol ratio of both thelayers were comparable (FIGS. 25 and 26). This suggests that oxidationis not restricted to the surfaces of the gel and that the diffusion ofH₂O₂ is rapid in this experimental set-up. However, if the gel is castedinto a 48-well plate and the H₂O₂ solution is merely applied over thetop of the gel (as per later experiments, see scheme in FIG. 31), thediffusion rate of H₂O₂ will be reduced as only the top surface isaccessible to the solution in this configuration.

Effects of Oxidation on G′ and Elasticity of the Gel

The rheological properties of various LK₆C gels were next quantified. Asa measure of stiffness, the elastic modulus, G′ was plotted againstangular frequency, m after the gel was subjected to 2-24 hours ofcast-and-soak oxidation with 0.06% H₂O₂, or 24 hours of in situoxidation with 0.03-0.1% H₂O₂ (FIG. 27 A). Compared to non-oxidisedgels, the stiffness was either maintained or increased followingdifferent oxidation regimes. Interestingly, the stiffness achieved after24 hours of cast-and-soak differed from that after in situ oxidationwith 0.06% H₂O₂. This could be due to the different availability of H₂O₂and sequence of oxidation. More specifically, in cast-and-soak, the gelhas access to a sink of H₂O₂ and cross-linking happens only afterself-assembly of peptide fibers; while during in situ oxidation, H₂O₂ islimited to the gel volume and cross-linking occurs concurrently withself-assembly. The linear viscoelastic (LVE) limit (elasticity) of thegel, however, increased 2.4-3.8 fold following oxidation (FIG. 27B).This is presumably attributed to the introduction of additional chemicalbonds. Other strategies to modulate the stiffness and elasticity of LK₆Cgels include varying its concentration (FIG. 29A/B) or doping LK₆C withLK6 (FIG. 29C/D). This also allows the tuning of the amount of thiolgroups available in the gel.

Fibrous Microstructures of Gels

Field emission scanning electron microscope (FESEM) revealed that thefibrous microstructures of LK₆C gels were maintained after variousoxidation regimes (FIG. 30).

H₂O₂ and Residual Acid Readily Removed

Using a configuration closer to cell culture experiments, gels werecasted overnight in 48-well plates and water was applied over the gelsto purify it before the introduction of cells. The majority of H₂O₂ andresidual acid can be removed be simply changing the water regularly(FIG. 31). Growth medium can be used instead of water to ensure that thegel is essentially free of H₂O₂ and adjusted to a pH amenable for cellculture.

Gradual and Tunable Release Kinetics

As a model system, dextran particles were encapsulated within the geland their release profile monitored. No burst release was observed inboth cases and release rate was suppressed in the oxidized sample,presumably due to the increased stability in water (FIG. 32).

Functionalization of the Hydrogel

The gel was next functionalized with a bioactive signal. CRGD (0-1mg/mL) was simply mixed with LK₆C (fixed at 10 mg/mL, i.e., CRGD liganddensity 0-9. 1 gross weight %) in the presence of H₂O₂ and ring-castedovernight. UPLC-MS confirmed the formation of LK₆C−CRGD conjugate andthe disappearance of free CRGD (FIG. 33). The mild and simple reactionconditions are to be noted. A person skilled in the art will alsorecognize the versatility of this peptide platform as well as the factthat future conjugations need not be limited to RGD. The gels were nextpurified by a dialysis-inspired method whereby water was layered on topand changed regularly. Doing so, >96% of unreacted H₂O₂ was removedafter 4 hrs and >99% after 7 hrs (FIG. 31 A). Similarly, >85% ofresidual acid from SPPS was removed after 8 hrs (FIG. 31 B).

Biocompatibility and Use for Cell Culture

To test for biocompatibility, HepG2 cells were either seeded directlyinto wells (2D control) or onto purified LK₆C gels conjugated withdifferent concentrations of RGD. After four days, calcein stainingrevealed that cells were viable in all experiments (FIG. 34 A/B). Thiswas repeated with primary rabbit fibroblasts (FIG. 35), 3T3 murinefibroblasts (FIG. 38) and NIH-3T3 murine fibroblasts (data not shown),confirming the biocompatibility of the gels. With regular media changes,cells remained viable for at least 3 weeks (FIG. 38). Images obtainedfrom the gel cultures were presented as mergers of several z-stacks.While that may already suggest a 3D distribution, the inventors wantedto verify that they were not artifacts due to, e.g., the meniscus on gelsurfaces or the gel being casted in a non-horizontal position. The gelswere therefore embedded vertically and cross-sectional slices wereobtained for depth profiling. From FIG. 34B, calcein-stained cells wereobserved to infiltrate the gel, resulting in multi-layered growth andvalidating their 3D spatial distribution. As visually suggested in FIG.34A, HepG2 cells appeared to have proliferated faster within gels withRGD compared to the ones without.

This was supported by data from the MTT assay (FIG. 34C). Being ananchorage-dependent cell line, the better adhesion to RGD conjugatedgels presumably provided the cells with a more ideal growth environment.Higher magnification images (FIG. 36) were subsequently taken and theaverage cell-spread area of HepG2 cells was quantified. Interestingly,3D gel culture had insignificant effects (p>0.05) on the averagecell-spread area of HepG2 cells compared to those in regular 2D culture(FIG. 34D). The amount of RGD ligand present also did not significantly(p>0.05) impact the cell spreading area of HepG2 cells. The trend wasdifferent, though, in the case of primary rabbit fibroblasts (FIG. 35B)and NIH-3T3 murine fibroblasts (data not shown), both of which spreadedmore in 2D culture. Cells are known to respond to mechanical propertiessuch as stiffness and elasticity of their microenvironment and theseobservations are consistent with an earlier report thathydrogel-encapsulated murine fibroblasts maintained roundermorphologies. However, while a transition from 2D to 3D culture causedthe fibroblasts to spread differently, the amount of RGD ligand presenthad insignificant effects (p>0.05). Compared to before CRGD attachment,the gel was as stiff (FIG. 37 A) but less elastic (FIG. 37B) afterconjugation.

Non-Allergenicity and Non-Toxicity

Experiments conducted by the CRO Toxikon further showed that LK₆C wasnon-allergenic and non-toxic. More particularly, LK₆C caused nosensitization on the skins of guinea pigs in the direct contact Kligmanmaximization test during a GLP study conducted by Toxikon according tothe ISO 10993-10 guidelines. Also, LK₆C exhibited no significanttoxicity in the direct contact V79 colony assay in another GLP studyperformed by Toxikon.

Use in Wound Treatment

A first round of wound healing experiments with mice was conducted. Inthis model, the epidermis and dermis of mice we removed to simulateinjury (FIG. 39). The following groups were analyzed:

a) No treatment control (i.e., simply bandage up the wound)

b) Application of LK₆C−CRGD gel

c) Application of LK₆C−CRGD gel with 3T3 murine fibroblasts cultured in3D

Here, the fibroblasts (the major population of the dermis) act as atherapeutic/bioactive agent. The hypothesis is that the 3T3 cellssecrete factors that encourage the growth of keratinocytes, which makeup the epidermis. This should further aid healing as compared to thegel-only treatment (group b). After 2 weeks, the extent ofvascularisation of the wound (a prerequisite feature of recovery) wasindeed most significant in group c, followed by group b and then group a(FIG. 40). Subsequent histology also showed the regeneration of asignificant dermal layer in group c mice. In comparison, group b micehad less regenerated dermis, and group a mice had the least extent ofdermal regeneration.

Summary

Cysteine-mediated disulfide-crosslinked ultra small peptide hydrogelwere analyzed. Cross-linking was driven by H₂O₂ and produced only wateras a by-product. Moreover, H₂O₂ helped to maintain the sterility of thegel and can be virtually removed before the introduction of cells.Oxidation increased both the elasticity of the gel and its ability tokeep its shape after being soaked in water. Due to the cysteine residue,bioactive signals can be conjugated to the peptide fibers using facilechemistry and gels can be easily purified. Gels formed were shown tosupport the true 3D distribution of cells and influence their growth andspreading characteristics. Furthermore, their applicability in woundtreatment was demonstrated.

The listing or discussion of a previously published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge. All documents listed are hereby incorporated herein byreference in their entirety for all purposes.

Exemplary embodiments of the invention illustratively described hereinmay suitably be practiced in the absence of any element or elements,limitation or limitations, not specifically disclosed herein. Thus, forexample, the terms “comprising”, “including”, “containing”, etc. shallbe read expansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by exemplary embodiments and optionalfeatures, modification and variation of the inventions embodied thereinherein disclosed maybe resorted to by those skilled in the art, and thatsuch modifications and variations are considered to be within the scopeof this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub generic groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention claimed is:
 1. A method of preparing a cell culturesubstrate, preparing a device for drug or gene delivery, or preparing awound dressing or implant that gels in situ, the method comprising:preparing a hydrogel by a method comprising steps of: dissolvingamphiphilic peptides having the general formula:Z_(p)—(X)_(n)—(Y)_(m)-AA_(thiol)-Z′_(q), wherein Z is an N-terminalprotecting group, X is, at each occurrence, independently selected froman aliphatic amino acid, Y is, at each occurrence, independentlyselected from a hydrophilic amino acid, AA_(thiol) is an amino acidcomprising a thiol group, Z′ is a C-terminal protecting group, n is aninteger selected from 2 to 6, m is selected from 0, 1 and 2, and p and qare independently selected from 0 and 1, in an aqueous solution, whereinthe aqueous solution comprises an oxidizing agent or wherein the methodfurther comprises the step of exposing the ready-made hydrogel to asolution of an oxidizing agent, forming the hydrogel into the cellculture substrate, the device for drug or gene delivery, or the wounddressing or implant that gels in situ.
 2. The method of claim 1, whereinthe amino acid comprising a thiol group is selected from cysteine andhomocysteine.
 3. The method of claim 1, wherein the oxidizing agent isH₂O₂.
 4. The method of claim 1, wherein the amphiphilic peptides aredissolved at a concentration from 0.01 μg/ml to 50 mg/ml; and optionallywherein the dissolved amphiphilic peptides in aqueous solution arefurther exposed to a temperature in the range of from 20° C. to 90° C.;and optionally wherein the dissolved amphiphilic peptides in aqueoussolution are exposed to the temperature for at least 1 hour.
 5. Themethod of claim 1, wherein the method further comprises a step of:exposing the ready-made hydrogel to an aqueous solution not comprisingthe oxidizing agent, wherein, if the method comprises the step ofexposing the ready-made hydrogel to a solution of the oxidizing agent,the step of exposing the ready-made hydrogel to an aqueous solution notcomprising the oxidizing agent is performed after the step of exposingthe ready-made hydrogel to a solution of the oxidizing agent.
 6. Themethod of claim 5, wherein the step of exposing the ready-made hydrogelto an aqueous solution not comprising the oxidizing agent is repeated atleast once; and optionally wherein the step of exposing the ready-madehydrogel to an aqueous solution not comprising the oxidizing agentoccurs for at least 1 hour; and optionally wherein the step of exposingthe ready-made hydrogel to an aqueous solution not comprising theoxidizing agent occurs at a temperature in the range of from 30° C. to45° C.
 7. The method of claim 1, wherein the method comprises at leastone of step of: adding at least one of a microorganism, a cell, a virusparticle, a peptide, a peptoid, a protein, a nucleic acid, anoligosaccharide, a polysaccharide, a vitamin, an inorganic molecule, anano- or microparticle, a synthetic polymer, a small organic molecule, acosmetic agent or a pharmaceutically active compound; adding at leastone non-peptidic polymer; adding at least one gelation enhancer; oradding at least one buffer.
 8. The method of claim 7, wherein thegelation enhancer is a salt or a solution of a salt.
 9. The method ofclaim 1, wherein the N-terminal protecting group is any of a group ofthe general formula —C(O)—R, wherein R is selected from the groupconsisting of H, unsubstituted or substituted alkyls, and unsubstitutedor substituted aryls; or a peptidomimetic molecule, including naturaland synthetic amino acid derivatives, wherein the N-terminus of thepeptidomimetic molecule may be modified with a functional group selectedfrom the group consisting of carboxylic acid, amide, alcohol, aldehyde,amine, imine, nitrile, an urea analog, thiol, phosphate, carbonate,sulfate, nitrate, maleimide, vinyl sulfone, azide, alkyne, alkene,carbohydrate, imide, peroxide, ester, thioester, aryl, ketone, sulphite,nitrite, phosphonate and silane.
 10. The method of claim 1, wherein theC-terminal protecting group is any one of an amide group or an estergroup; or a peptidomimetic molecule, including natural and syntheticamino acid derivatives, wherein the C-terminus of the peptidomimeticmolecule may be modified with a functional group selected from the groupconsisting of carboxylic acid, amide, alcohol, aldehyde, amine, imine,nitrile, an urea analog, thiol, phosphate, carbonate, sulfate, nitrate,maleimide, vinyl sulfone, azide, alkyne, alkene, carbohydrate, imide,peroxide, ester, thioester, aryl, ketone, sulphite, nitrite, phosphonateand silane.
 11. The method of claim 1, wherein, for a given amphiphilicpeptide, the aliphatic amino acid, the hydrophilic amino acid and theamino acid comprising a thiol group are either D-amino acids or L-aminoacids.
 12. The method of claim 1, wherein the hydrophilic amino acid hasa polar group which is independently selected from a hydroxyl, an ether,a carboxyl, an imido, an amido, an ester, an amino, a guanidino, a thio,a thioether, a seleno, and a telluro group.
 13. The method of claim 1,wherein the aliphatic amino acid is selected from the group consistingof isoleucine, norleucine, leucine, valine, alanine, glycine,homoallylglycine and homopropargylglycine.
 14. The method of claim 1,wherein all or a portion of the aliphatic amino acids of the amphiphilicpeptides are arranged in an order of decreasing amino acid size in thedirection from N- to C-terminus of the amphiphilic peptides, wherein thesize of the aliphatic amino acids is defined as I=L>V>A>G.
 15. Themethod of claim 1, wherein the aliphatic amino acids arranged in anorder of decreasing amino acid size have a sequence selected from LIVAG(SEQ ID NO. 54), ILVAG (SEQ ID NO. 55), LIVAA (SEQ ID NO. 56), LAVAG(SEQ ID NO. 57), IVAG (SEQ ID NO. 58), LIVA (SEQ ID NO. 59), LIVG (SEQID NO. 60), IVA and IV, wherein, optionally, there is an A precedingsuch sequence at the N-terminus.
 16. The method of claim 1, wherein theamphiphilic peptides are the same or different.
 17. The method of claim1, wherein (X)_(n)-(Y)_(m)-AA_(thiol) is selected from the groupconsisting of LIVAGKC (SEQ ID NO: 43), LIVAGSC (SEQ ID NO: 44), LIVAGDC(SEQ ID NO: 45), ILVAGKC (SEQ ID NO: 46), ILVAGDC (SEQ ID NO: 47),LIVAGC (SEQ ID NO: 48), AIVAGC (SEQ ID NO: 49), ILVAGC (SEQ ID NO: 50),IVKC (SEQ ID NO: 51), IVDC (SEQ ID NO: 52) and IVSC (SEQ ID NO: 53). 18.The method of claim 1, wherein at least 5% of the plurality ofamphiphilic peptides are chemically cross-linked.